Synthesis and use of isotopically-labelled glycans

ABSTRACT

Isotopically-labelled glycans and their synthesis and use as internal standards in the analysis by mass spectrometry of glycan mixtures is described. The methods of synthesis described herein may be used conveniently to prepare libraries of heavy glycans for use in the qualitative and quantitative identification of glycans in natural samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of International Application No.PCT/EP2014/056737, filed Apr. 3, 2014, which claims priority from GreatBritain Patent Application No. 1305986.0, filed Apr. 3, 2013. The entiredisclosure of each of the aforesaid applications is incorporated byreference in the present application.

FIELD OF THE INVENTION

The present invention relates to isotopologues of oligosaccharides andpolysaccharides. In particular, the present invention relates to thesynthesis of isotopically-labelled glycans and their use as standards inthe analysis by mass spectrometry of glycan mixtures.

BACKGROUND OF THE INVENTION

Glycosylation is one of the most common post-translational proteinmodifications in eukaryotic systems. It has been estimated that overhalf of all mammalian proteins are glycosylated at some point duringtheir existence and virtually all membrane and secreted proteins areglycosylated. Glycosylation is a non-template-driven process and isbelieved to introduce the high level of variability necessary forcomplex processes in higher organisms. In addition to participating inkey macromolecular interactions, glycans have been shown to contributeto protein folding, trafficking, and stability.

N-glycans are linked to the protein backbone via asparagine residuesthat are part of the tripeptide sequences Asn-X-Ser or Asn-X-Thr, with Xbeing any amino acid except proline. Depending on the terminal sugarresidues, N-glycans are classified into complex, high-mannose, andhybrid N-glycans. This classification is based on the commonpentasaccharide motif shared by most N-glycans. O-glycans are linked viaserine or threonine residues to the protein. There are a number ofO-glycan core structures, with the most common being Core 1, Core 2,Core 3, and Core 4.

Numerous diseases are known to involve acquired changes in glycosylationand/or in the recognition of glycans. For example, altered glycosylationis a universal feature of cancer cells and some glycan structures arewell-known markers for tumours and tumour progression. As a result,methods for the comprehensive analysis of protein glycosylation andglycan composition are of interest to the scientific community.

For most glycol-profiling methods, the glycans are removed from theprotein either by hydrazinolysis or treatment with a specific peptideglycosidase (e.g. PNGase F). Owing to its high sensitivity at lowconcentrations, mass spectrometry is often used in the analysis of theresulting complex mixtures. However, the signal intensity of particularanalytes is dependent, amongst many other factors, on the physicalproperties (likelihood of ionisation, tendency to fragment, etc.) of theanalyte, making any relative quantification, and sometimes evenidentification, very difficult.

Identification of glycans common to two samples and their relativequantification may be facilitated by use of derivatisation of the glycanmixtures to incorporate isotopic tags. The two samples are labelled withthe light and heavy form of the labelling reagent and then mixed priorto analysis using mass spectrometry. Derivatisation to incorporateisotopic tags into glycan mixtures isolated from glycoproteins has beenaccomplished using permethylation techniques or glycan reductive isotopelabelling, in which the tag is introduced using reductive amination(Atwood, 2007; Bowman, 2007, 2010; Botelho, 2008; Hitchcock, 2006; Hsu,2006; Kang, 2007; Lawrence, 2008; Ridlova, 2008; Yuan, 2005; Zhang,2003).

Reductive amination typically occurs at the reducing end of the glycansand may use isotopically-labelled aniline, aminopyridine or anthranilicacid. For example, Xia et al. (2009) have demonstrated the use ofisotopically-labelled aniline tags to compare the differences inmixtures of glycans released from human and mouse sera. The glycans werereleased by PNGase F, then the resulting mixtures separately derivatisedby reductive amination with ¹²C₆-aniline or ¹³C₆-aniline. By analysingan equimolar combination of the ¹²C₆-aniline-derivatised mixture ofglycans from mouse serum and the ¹³C₆-aniline-derivatised mixture ofglycans from human serum, the authors reported that they were able toidentify paired mass peaks separated by a mass difference of 6 Da andassign plausible structures for glycans common to both samples. Theauthors reported that a comparison of the relative intensities of thesepeaks enabled a determination of the amount of a particular commonglycan present in one sample compared to the other.

However, these methods provide only semi-quantitative results.Furthermore, the results are affected by the reproducibility of thetagging procedures and problems caused by side reactions, oxidativedegradation and “peeling reactions” (which may occur due to certainreaction conditions in aqueous solutions), and importantfunctionalization may be lost during the derivatisation step.

Isotopic tags have also been used in proteomics. Breidenbach et al.(2012) have demonstrated the metabolic incorporation ofisotopically-labelled GlcNAc into yeast N-glycans using filter aidedsample preparation methodology. A GlcNAc isomix was used comprisingnatural isotope abundance GlcNAc, ¹³C₂-GlcNAc and ¹³C₄ ¹⁵N₁-GlcNAc in a1:2:1 ratio to mimic the dibromide isotope triplet pattern. Theresulting glycol conjugates containing the isomix underwent FASPdigestion and EndoH deglycosylation and were analysed using an automatedisotopic envelope pattern search (in LC-MS/MS experiments) to facilitateglycoside identification. The method enabled the authors to placefragmentation priority on glycopeptides ions regardless of theirrelative intensities to other ions in the sample.

There exists an unmet need for improved methods for rapidly and easilyanalysing the content of released glycan mixtures, and in particular onewhich does not suffer from the disadvantages of the described prior art.

SUMMARY OF THE INVENTION

The present invention is based on the inventors' insight that stableisotopologues of individual glycans and glycoconjugates used asstandards in mass spectrometry may have utility in the qualitative andquantitative analysis of complex mixtures. In particular, the presentinvention addresses the problems of reproducibility and loss offunctional information associated with the isotopic glycan-taggingprocedures known in the art, in which glycan mixtures are derivatised toincorporate the tag either during or following removal from the protein.The present inventors have provided methods for the synthetic generationof isotopically-labelled glycans then may then be doped into an analytesample and analysed by mass spectrometry. These methods allow for theidentification of glycans of known structure in analyte sample throughcomparison of the mass spectrometry envelope(s) associated with theremaining mass spectrometry peaks (associated with the sample).

Furthermore, the present invention allows quantification of particularglycans within the sample in absolute terms through addition of a knownamount of the standard, representing a significant advantage over themethods known in the art which provide only semi-quantitative data. Thepresent invention provides libraries of isotopically-labelled glycanstandards (so-called “tagged standards”) for use in the qualitative andquantitative analysis of complex glycan mixtures using massspectrometry. Further provided are methods for using these standards toanalyse qualitatively and/or quantitatively the composition of complexglycan mixtures. These methods of analysis may have utility in theidentification of glycan markers associated with particular disordersand disease states and other biological processes.

Broadly, the present invention includes methods for the synthesis ofisotopically-labelled glycans (including glycoconjugates) comprisingtreating a glycan comprising at least two sugar units with anisotopically-labelled acylating agent to incorporate isotopic labelsinto glycan structures.

These isotopically-labelled oligosaccharides are oligosaccharide corestructures which may then be used to synthesise libraries ofisotopically-labelled glycan standards through enzymatic derivatisationsteps, that is, diversification by one or more enzyme-catalysed steps.This represents a significant advance in methods for the qualitative(and quantitative) detection of particular glycans.

Accordingly, in a first aspect, the present invention relates to amethod for the synthesis of an isotopically-labelled oligosaccharide,the method comprising:

-   -   acylating an oligosaccharide with an isotopically-labelled        acylating agent, wherein the oligosaccharide is optionally        protected with one or more protecting groups.

In a first aspect, the present invention provides a method for thesynthesis of an isotopically-labelled glycan for use as a massspectrometry internal standard, the method comprising:

-   -   acylating an oligosaccharide core structure with an        isotopically-labelled acylating agent, wherein the        oligosaccharide core structure is optionally protected with one        or more protecting groups, to obtain an isotopically-labelled        oligosaccharide core structure; and    -   enzymatically derivatising the resultant isotopically-labelled        oligosaccharide to obtain the isotopically-labelled glycan.

Enzymatic derivatisation, which may also be referred to as enzymaticdiversification, as used herein, refers to subjecting an oligosaccharideas described herein to an enzyme-catalysed reaction. Suitable enzymaticderivitisation/diversification reactions include:

-   -   Elongation: the addition of (a) further sugar unit(s) to the        oligosaccharide, typically via a condensation reaction with a        suitable sugar donor using a glycosyltransferase. Elongation may        occur at a terminus of the oligosaccharide, or on an        intermediate sugar unit.    -   Truncation: the removal of (a) sugar unit(s) from a terminus of        the oligosaccharide. This is typically via a hydrolysis reaction        with a hydrolase.    -   Epimerisation: the total or partial inversion of a stereocentre        in the molecule. This is typically catalysed by an epimerase.    -   Transglycosylation (glycosyl transfer reactions): the transfer        of a sugar unit from a donor to an acceptor; that is, one        oligosaccharide loses a sugar unit, i.e. is truncated, while        another oligosaccharide gains a sugar unit (i.e. is elongated).        These reactions may be catalysed by a synthetase or        transglycosidase.    -   Post-translation modification: other functionalization may be        incorporated during enzymatic derivatisation. Common        post-translational modifications are known in the art, and may        include, without limitation, phosphorylation, sulfation and        acylation.

Typically, such enzyme-catalysed derivatisation steps arechemo-selective. It will be appreciated that the first aspect of thepresent invention provides methods for the convenient generation of vastnumbers of isotopically-labelled glycan structures for use as massspectrometry internal standards starting from relatively small “core”oligosaccharides, as described herein.

In some embodiments, the enzymatic derivatisation step comprises a stepof enzymatic hydrolysis to remove a terminal sugar unit. This allowsaccess to asymmetric standards derived from, for example, thebiantennary heptasaccharide N-glycan core, synthesised as describedherein.

In some embodiments, the enzymatic derivatisation step comprises a stepof enzymatic elongation of the resultant glycan with aglycosyltransferase and a suitable sugar donor. Suitable methods ofenzymatic elongation are known in the art (Blixt, 2006; Ruiz, 2001;Serna, 2010, Zou, 2011) and are further described below. Suitable sugardonors may be mono-, oligo- or poly-saccharides. In some embodiments,the step of enzymatic elongation is repeated one or more times. Inembodiments in which the step of enzymatic elongation is repeated,preferably the sugar donor in each step is a suitable monosaccharidesugar donor.

In some embodiments, the sugar donor in the enzymatic elongation step isisotopically-labelled. In embodiments in which the step of enzymaticelongation is repeated, the sugar donor in an enzymatic elongation stepmay be isotopically-labelled independent of whether or not the sugardonor in any other enzymatic elongation step is or is notisotopically-labelled. The sugar donor may be isotopically-labelled withany suitable isotopic form of an atom at any suitable position. In thisway, additional isotopically-labelled monosaccharide units may beincorporated into the isotopically-labelled oligosaccharide. This mayhave utility in the analysis of glycans using fragmentation patternsobtained in MS-MS methods and for generating different isotopologues ofa particular glycan structure to aid accurate quantitative analysis asdescribed below.

It will be appreciated that the enzymatic derivatisation step maycomprise a single enzyme-catalysed step, or may include more than oneenzyme-catalysed step in sequence. For example, the enzymaticderivatisation step may be a single hydrolysis or elongation step, ormay include more than more one hydrolysis and/or elongation step togenerate the desired glycan structures. For example, anisotopically-labelled oligosaccharide core may be sequentiallyelongated, or truncated and then (sequentially) elongated. Anisotopically-labelled oligosaccharide may also be elongated and thentruncated at a different position. Different ordering of the steps maybe desirable to suit the specificity of the enzymes used. Representativenon-limiting examples are provided herein.

As used herein, the term oligosaccharide pertains to saccharide polymerscomprising at least two simple sugars (monosaccharide units). In someembodiments, the oligosaccharide is a disaccharide. In some embodiments,the oligosaccharide is a trisaccharide. In some embodiments, theoligosaccharide is a tetrasaccharide. In some embodiments, theoligosaccharide is a pentasaccharide. In some embodiments, theoligosaccharide is a hexasaccharide. In some embodiments, theoligosaccharide is a heptasaccharide. In some embodiments, theoligosaccharide is a higher oligomer. The oligosaccharide may be linearor branched (also referred to as antennary).

The oligosaccharide may comprise one or more hydroxyl or amino groups,each of which may independently be protected with a protecting group. Insome embodiments, free hydroxyl and/or amino groups are not protected.In other embodiments, some or all of the hydroxyl groups present areprotected. In some embodiments, at least one protected primary and/orsecondary amino group is present.

Suitable protecting groups for hydroxyl and amino groups are known inthe art. Purely by way of example, and without limitation, suitableprotecting groups for use in the present invention are discussed below.In some embodiments, protecting groups are selected to be orthogonal toeach other to facilitate selective deprotection and chemicalmanipulation at desired positions on the oligosaccharide.

Preferably, the oligosaccharide comprises at least one free —NH₂ groupand acylation occurs at the free —NH₂ group. Where more than one free—NH₂ group is present, acylation may occur at each free —NH₂ group.

The present inventors have found that semi-protected core motifs aresuitable substrates for chemoselective enzymatic derivatisation.Furthermore, the presence of the protecting groups may have particularadvantages. For example, as described herein, benzylic groups may act aschromophores for peak detection during HPLC analysis and purification,and may aid separation of different products, for example, isomericglycans.

Accordingly, in some methods described herein, the isotopically-labelledcore oligosaccharides are partially protected during the enzymaticderivatisation step. Suitably, the protecting groups are optionallysubstituted benzyl groups, preferably —CH₂Ph groups, present on one ormore hydroxyl groups.

For example, the methods described herein may include at least oneenzymatic derivatisation step on an isotopically-labelled partiallyprotected oligosaccharide, followed by a deprotection step. Furtherenzymatic derivatisation step(s) may then follow. For example, thepartially protected oligosaccharide may be partially benzylated.Suitably, when the oligosaccharide has one or more benzyl groups, thedeprotection step may be hydrogenation.

In some embodiments, the oligosaccharide comprises a disaccharide motif,the disaccharide motif comprising a first monosaccharide unit and asecond monosaccharide unit, wherein at least one of the firstmonosaccharide unit and/or second monosaccharide unit comprises an aminogroup and acylation occurs at the amino group(s). In embodiments inwhich the oligosaccharide comprises more than two monosaccharide units,the disaccharide motif may be located at a terminus of the saccharidechain, which may be a reducing or non-reducing end. In embodiments inwhich the oligosaccharide comprises more than four monosaccharide units,the disaccharide motif may be located at a terminus or an intermediateposition within the saccharide chain.

The monosaccharide comprising an amino group is preferably an aminosugar monosaccharide. Broadly, any amino sugar monosaccharide having atleast one —NH₂ group may be suitable as at least one of the firstmonosaccharide unit and/or second monosaccharide unit in methods of thefirst aspect of the present invention. Examples of suitable amino sugarsinclude, but are not limited to, hexosamines and derivatives thereof.Examples of suitable amino sugar monosaccharidees include, but are notlimited to, glucosamine (GlcN), galactosamine (GalN), mannosamine(ManN), fructosamine (FruN), fucosamine (FucN), muramic acid (Mur),neuraminic acid (Neu), daunosamine, and perosamine.

Other amino sugars derivatives may also be suitable for use according tothe present invention. Accordingly, in some embodiments, the firstmonosaccharide unit and/or second monosaccharide unit is a des-acetylderivative of an N-acetyl amino sugar. Suitable examples, in addition tothose listed above, include, for example, des-acetylaspartyl-glucosamine. Monosaccharide units may also be furthersubstituted and comprise a part of, for example, a glycoside.

The second monosaccharide unit may be a second amino sugar, an acetylamino sugar, or other sugar unit. For example, and not by way oflimitation, the second monosaccharide unit may be a hexose or pentose oramino sugar thereof, and may be further substituted with, for example,fatty acid chains (for example, the second monosaccharide unit may belipid A).

In some embodiments in which the oligosaccharide comprises adisaccharide motif, the first monosaccharide unit is selected from:

-   -   GlcN, GalN, ManN, FruN, FucN, Mur, or Neu;

and the second monosaccharide unit is selected from:

-   -   Glc, Gal, Man, Rha, Fru, Fuc, GlcN, GalN, ManN, FruN, FucN, Mur,        Neu, GlcNAc, GalNAc, ManNAc, FruNAc, FucNAc, MurNAc, NeuNAc,        sialic acid, or inositol.

The first monosaccharide sugar and the second monosaccharide sugar unitmay be arranged in the sequences, from reducing end to non-reducing end,first monosaccharide unit followed by second monosaccharide unit, orsecond monosaccharide unit followed by first monosaccharide unit.

In one embodiment, the sequence is:

-   -   first monosaccharide-second monosaccharide.

In one embodiment, the sequence is:

-   -   second monosaccharide-first monosaccharide.

In some preferred embodiments, the first and second monosaccharide unitsof the disaccharide motif of the oligosaccharide are selected from themonosaccharide units associated with N- and O-glycan cores anddes-acetyl forms thereof. Accordingly, in some embodiments, the firstmonosaccharide unit is glucosamine or galactosamine and the secondmonosaccharide unit is selected from mannose, galactose, glucosamine,galactosamine, N-acetyl-glucosamine, or N-acetyl-galactosamine. In someembodiments, the first monosaccharide unit is glucosamine and the secondmonosaccharide unit is selected from mannose, galactose, glucosamine,galactosamine, N-acetyl-glucosamine, or N-acetyl-galactosamine,preferably from mannose or glucosamine. In some embodiments, the firstmonosaccharide unit is galactosamine and the second monosaccharide unitis selected from mannose, galactose, glucosamine, galactosamine,N-acetyl-glucosamine, or N-acetyl-galactosamine, preferably from mannoseor glucosamine.

In some embodiments, the method comprises reacting an oligosaccharidecomprising a motif selected from:

-   -   GlcN-GlcN    -   Gal-GalN    -   GalN-GlcN

with an isotopically-labelled acylating agent. Further monosaccharideunits may be present at the reducing and/or non-reducing ends.

In some embodiments, the method comprises reacting an oligosaccharidecomprising the motif:

-   -   Man-GlcN-GlcN

with an isotopically-labelled acylating agent. Further monosaccharideunits may be present at the reducing and/or non-reducing ends.

In a preferred embodiment, the method comprises reacting anoligosaccharide comprising the motif:

-   -   Man-GlcN-GlcN

with an isotopically-labelled acetylating agent to obtain anoligosaccharide comprising the motif:

-   -   Man-GlcNAc*-GlcNAc*

wherein Ac* is an isotopically-labelled acetyl group.

In some embodiments, the method comprises reacting an oligosaccharide offormula (A) with an isotopically-labelled acetylating agent underconditions suitable to form an oligosaccharide of formula (B):

wherein:

each R¹ is independently H or a protecting group;

R² is independently H or a protecting group;

each R³ independently is H, a protecting group, or (Sac), wherein each

Sac is a monosaccharide unit and m is a number between 1 and 50.

In some embodiments, the method comprises reacting an oligosaccharide offormula (C) with an isotopically-labelled acetylating agent underconditions suitable to form an oligosaccharide of formula (D):

wherein:

each R¹ is independently H or a protecting group;

R² is independently H or a protecting group;

each R³ is independently H, a protecting group, or (Sac)_(m) whereineach Sac is a monosaccharide unit and m is a number between 1 and 50.

As defined above, in some embodiments, each R¹, R² and R³ mayindependently be a protecting group. The protecting groups may be thesame or different. For example, if more than one R³ is a protectinggroup, each R³ may be the same as, or different to, any other protectinggroup. In some embodiments, at least one R³ is (Sac)_(m), wherein m is anumber between 1 and 50. In embodiments in which more than one R³ is(Sac)_(m), each (Sac)_(m) may independently be the same or different toany other (Sac)_(m) in the molecule.

In some embodiments, m is a number between 1 and 20.

In some embodiments, m is a number between 1 and 10.

In some embodiments, m is a number between 1 and 5.

In some embodiments, m is 1 or 2.

In some embodiments, R² is H. In some embodiments, each R³ is H. In apreferred embodiment, each R¹ is benzyl, R² is H, and each R³ is H.

In some embodiments, the method comprises:

glycosylating an oligosaccharide of formula (I):

wherein each of P¹, P², P³, P⁴, and P⁵ is independently a protectinggroup, or optionally P⁴ and P⁵ together form an acetal group;

with a sugar donor of general formula (II):(Sac)_(n)-Sac-LG  (II)wherein each Sac is a monosaccharide unit, n is a number between 0 and50, and -LG represents the non-glycosylated anomeric position of thedonor-sugar primed with a suitable leaving group;

to give an oligosaccharide of formula (III):

removing P⁴ to reveal a hydroxyl group and glycosylating the resultinghydroxyl group with a sugar donor of general formula (II);

removing each P³ group to reveal a free amino group and acetylating eachresultant free amino group with an isotopically-labelled acetylatingagent.

In some embodiments, n is a number between 0 and 20.

In some embodiments, n is a number between 0 and 10.

In some embodiments, n is a number between 0 and 5.

In some embodiments, n is 0 or 1.

Suitable sugar donors are known in the art and may include, withoutlimitation, glycosyl halides, for example, glycosyl fluorides andbromides; glycosyl phosphates, glycosyl trihaloacetimidates, n-pentenylglycosides (and more generally, suitable hemiacetals, orthoesters and1-oxygen substituted glycosyl donors) and thio-glycosides. Thereactivity of a sugar donor may depend upon the nature of any protectinggroups present. Sugar donors may be disarmed (for example, by protectionwith acetyl groups), armed (for example, by protection with benzylgroups) or super-armed (for example, by protection with bulky silylgroups).

In some preferred embodiments, the sugar donor leaving group comprises atrihaloacetimidate group, preferably a trifluoroacetimidate group, suchas, for example:

In some embodiments, the method comprises the step of glycosylating theC₂-position of monosaccharide unit A to obtain a bi-antennary glycan. Insome embodiments, the method comprises the step of glycosylating theC4-position of monosaccharide unit A. Glycosylation at the C4-positionmay follow glycosylation at the C2-position to yield a tri-antennaryglycan, or may occur without a prior C2-glycosylation step to yield abi-antennary glycan. In embodiments in which the method comprisesglycosylation at both C2 and C4 of monosaccharide unit A, theglycosylation steps may occur in either order.

Chemo-selective glycosylation may be enzymatically-catalysed and/or maybe facilitated by selective protection and/or protecting groupstrategies.

Preferably, the oligosaccharide core used for the enzymaticderivatisation is comprises 3 to 9 monosaccharide units.

In some embodiments of the methods of the present invention, theisotopically-labelled acylating agent may be an acyl halide or ananhydride of a suitable carboxylic acid. In some preferred embodiments,the isotopically-labelled acylating agent is isotopically-labelledacetic anhydride, preferably (¹³CH₃ ¹³C═O)₂, (¹³CH₃C═O)₂, (CH₃ ¹³C═O)₂,(CD₃C═O)₂, (¹³CD₃ ¹³C═O)₂, (¹³CD₃C═O)₂, or (CD₃ ¹³C═O)₂. In someembodiments, the isotopically-labelled acylating agent is (¹³CH₃¹³C═O)₂.

In some embodiments of the present invention, each Ac*, if present, isselected from —(¹³C═O)¹³CH₃, —(C═O)¹³CH₃, —(¹³C═O)CH₃, —(C═O)CD₃,—(¹³C═O)¹³CD₃, —(C═O)¹³CD₃, —(¹³C═O)CD₃, —(¹⁴C═O)¹⁴CH₃, —(C═O)¹⁴CH₃,—(¹⁴C═O)CH₃, —(C═¹⁷O) CH₃, —(¹³C═¹⁷O) CH₃, —(C═¹⁷O)¹³CH₃,—(¹³C═¹⁷O)¹³CH₃, —(C═¹⁸O)CH₃, —(¹³C═¹⁸O) CH₃, —(C═¹⁸O)¹³CH₃,—(¹³C═¹⁸O)¹³CH₃.

In some embodiments of the present invention, each Ac*, if present, isselected from —(¹³C═O)¹³CH₃, —(C═O)¹³CH₃, —(¹³C═O)CH₃, —(C═O)CD₃,—(¹³C═O)¹³CD₃, —(C═O)¹³CD₃, —(¹³C═O)CD₃, —(¹⁴C═O)¹⁴CH₃, —(C═O)¹⁴CH₃,—(¹⁴C═O)CH₃, —(C═¹⁷O) CH₃, or —(C═¹⁸O)CH₃.

In some embodiments of the present invention, each Ac*, if present, isselected from —(¹³C═O)¹³CH₃, —(C═O)¹³CH₃, —(¹³C═O)CH₃, —(C═O)CD₃,—(¹³C═O)¹³CD₃, —(C═O)¹³CD₃ or —(¹³C═O)CD₃. In some embodiments thepresent invention, each Ac*, if present, is selected from —(¹³C═O)¹³CH₃,—(C═O)¹³CH₃, or —(¹³C═O)CH₃. In some embodiments of the methods, eachAc*, if present, is —(¹³C═O)¹³CH₃.

In some embodiments, methods according to the first aspect furthercomprise forming an oxazoline at a free anomeric position of anacetyl-hexosamine unit in the isotopically-labelled oligosaccharide. Theresultant isotopically-labelled glycan oxazoline may then be used toprepare an isotopically-labelled glycoconjugate.

In some embodiments, methods according to the first aspect furthercomprise glycosylating a peptide, lipid or protein to obtain anisotopically-labelled glycopeptide, peptidoglycan, glycolipid,glycoprotein comprising the isotopically-labelled oligosaccharide.

In a further aspect, the present invention provides anisotopically-labelled oligosaccharide or glycoconjugate obtainable by amethod according to the first aspect.

In a further aspect, the present invention provides a glycan comprisinga motif selected from:

wherein each Ac* is isotopically-labelled. The term glycan, as usedherein, refers to any saccharide in free form or forming a carbohydrateportion of a glycoconjugate.

In some embodiments, the motif is:Man-GlcNAc*-GlcNAc*.

wherein each Ac* is isotopically-labelled.

In some embodiments, the glycan comprises the motif:

wherein each Ac* is isotopically-labelled.

In some embodiments, each Ac* if present, is selected from—(¹³C═O)¹³CH₃, —(C═O)¹³CH₃, —(¹³C═O)CH₃, —(C═O)CD₃, —(¹³C═O)¹³CD₃,—(C═O)¹³CD₃, —(¹³C═O)CD₃, —(¹⁴C═O)¹⁴CH₃, —(C═O)¹⁴CH₃, —(¹⁴C═O)CH₃,—(C═¹⁷O)CH₃, or —(C═¹⁸O)CH₃

In some embodiments, the glycan comprises one or more furthermonosaccharide units, wherein each further monosaccharide, if present,may be independently isotopically-labelled.

In some embodiments, the number of further monosaccharide units isgreater than 10.

In some embodiments, the number of further monosaccharide units isgreater than 30.

In some embodiments, the number of further monosaccharide units isgreater than 50.

In some embodiments, the number of further monosaccharide units isgreater than 100.

In some embodiments, the present invention provides a glycan that hasthe structure:

This structure is an especially suitable core oligosaccharide substratefor enzymatic derivatisation to afford a variety ofisotopically-labelled N-glycans.

Where each Ac* is —(¹³C═O)¹³CH₃, this structure is referred to herein as¹³C₈-G0. As described herein, there may be advantages to using asemi-protected core oligosaccharide as a substrate for enzymaticderivatisation. Accordingly, in some embodiments, the five hydroxylmoieties at the GlcNAc*-GlcNAc* reducing end bear optionally-substitutedbenzyl groups. When each Ac* is —(¹³C═O)¹³CH₃ and these five hydroxyleach bear a PhCH₂— moiety, the structure is referred to herein as¹³C₈-G0(Bn₅).

In a further aspect, the present invention provides a method ofidentifying a glycan in a sample, the method comprising adding a taggedstandard comprising an isotopically-labelled glycan to the sample toobtain a doped sample, and analysing the doped sample using massspectrometry. In preferred embodiments, the isotopically-labelled glycanis an isotopically-labelled glycan according to the present inventionand/or obtainable as described herein.

Preferably, the tagged standard comprises an isotopically-labelledglycan that is an isotopologue of a glycan suspected to be present inthe sample. In some embodiments, the tagged standard comprises more thanone isotopically-labelled glycan. In some embodiments more than onetagged standard may be added to the sample to obtain the doped sample.In some embodiments, more than one isotopically-labelled glycan may beadded to facilitate simultaneous identification of multiple glycans in asample.

An advantage of methods of the present invention over methods known inthe prior art is that tagging of the glycan(s) in the sample toincorporate a tag is not necessary, avoiding issues of reproducibilityof tagging procedures and side reactions. Furthermore, sources ofexperimental variability which may be present in methods known in theart are cancelled out as both the isotopically-labelled glycan(s) (inthe tagged standard) and the analyte(s) are analysed in the sameexperiment and treated using the same procedures. Eachisotopically-labelled glycan ionises with the same efficiency as thecorresponding analyte but is easily identifiable by its fixed massincrement. In some embodiments, the tagged standard has a pre-determinedmass spectrometry spectrum, which may aid in the analysis of the dopedsample by enabling ion peaks associated with the isotopically-labelledglycan to be easily identified.

In preferred embodiments, a known amount of the isotopically-labelledglycan is added to the sample such that the glycan content of the samplecan be quantified by comparison of the relative intensity of the ionpeaks associated with the glycan and the isotopically-labelled glycan.Further details regarding the quantification of glycan content areprovided below. Accordingly, through the addition of a known amount ofthe isotopically-labelled glycan, an analyte may be quantified inabsolute terms even in a complex biofluid. In some embodiments, knownamounts of more than one isotopically-labelled glycan may be added tofacilitate simultaneous identification and quantification of multipleglycans in a sample.

In some embodiments, the method comprises:

(i) selecting a tagged standard comprising one or moreisotopically-labelled glycans;

(ii) adding the tagged standard to the sample to obtain a doped sample;

(iii) analysing the doped sample using mass spectrometry to obtain ionpeaks;

(iv) comparing the identity and intensity of the ion peaks associatedwith the tagged standard with the additional ion peaks in the spectrumof the doped sample.

In some preferred embodiments, the tagged standard is selected tocorrespond to the suspected glycan content of the sample. For example,and not by way of limitation, if a sample is suspected to comprise acombination of three glycan species, a tagged standard comprisingisotopologues of these three glycans may be selected.

The glycan(s) may be derivatised prior to analysing the doped sample.Derivatisation steps may include, for example, permethylation orderivatisation of sialic acid residues, if present, and may comprise aclean-up step. In some embodiments, the derivatisation comprisesglycosidase treatment for removal of sialic acids or other terminalsugar units.

In some preferred embodiments, the mass spectrometry is MALDI-ToF,direct infusion ESI-ToF or LC-MS, and may further comprise fragmentationby tandem mass spectrometry (sometimes called MS-MS), which mayfacilitate analyte identification and enable isobaric analytes to bedistinguished in complex mixtures. Fragmentation may be achieved using,for example, collision induced dissociation (CID), electron capturedissociation (ECD), electron transfer dissociation (ETD), infraredmultiphoton dissociation (IRMPD), black body infrared radiativedissociation (BIRD), electron-detachment dissociation (EDD) orsurface-induced dissociation (SID), or any other suitable method.

In some embodiments, the sample is a complex biofluid, and the glycan inthe sample may, for example, be a glycan released from a recombinantglycoprotein or antibody. The glycan in the sample may a biomarkerassociated with a medical disease or disorder, or a biological process,and in some preferred embodiments, the method further comprisescorrelating the presence or amount of one or more of the glycans as anindicator of a medical disease or disorder, or a biological process.Without limitation, the medical disease or disorder may be selected fromcancer, a cardiovascular disorder, an inflammatory skin disease,diabetes mellitus, a gastrointestinal disorder, a liver disorder,anaemia, an immunological disease or disorder, autoimmune disease,arthritis, including rheumatoid arthritis, an infectious disease,nephropathy, a neurological disorder, a pulmonary disorder or acongenital disorder of glycosylation.

These methods may be performed in vitro.

Accordingly, in a further aspect, the present invention provides amethod for diagnosing a patient suspected of having a disease associatedwith a glycan, the method comprising:

-   -   (i) obtaining a sample suspected of containing the glycan;    -   (ii) selecting a tagged standard comprising an        isotopically-labelled glycan corresponding to the glycan        associated with the disease;    -   (iii) adding the tagged standard to the sample to obtain a doped        sample;    -   (iv) analysing the doped sample using mass spectrometry to        obtain ion peaks;    -   (v) comparing the identity and intensity of the ion peaks        associated with the tagged standard with the additional ion        peaks in the spectrum of the doped sample;    -   (vi) using the presence of said glycan to assist diagnosis of        the disease or disorder.

In a further aspect, the present invention provides anisotopically-labelled glycan as described herein for use in a method ofdiagnosis, the method comprising:

-   -   (i) obtaining a sample suspected of containing a glycan        associated with a disease or disorder from a patient;    -   (ii) selecting a tagged standard comprising an        isotopically-labelled glycan corresponding to the glycan        associated with the disease or disorder;    -   (iii) adding the tagged standard to the sample to obtain a doped        sample;    -   (iv) analysing the doped sample using mass spectrometry to        obtain ion peaks;    -   (v) comparing the identity and intensity of the ion peaks        associated with the tagged standard with the additional ion        peaks in the spectrum of the doped sample to identify, and        optionally to quantify, the presence of one or more glycans in        the sample;    -   (vi) using the presence of said one or more glycans to diagnose        the disease or disorder.

In further aspects, the present invention provides anisotopically-labelled glycan as described herein for use in a method ofdiagnosis, and methods of diagnosis, the method comprising:

-   -   (i) selecting a tagged standard comprising an        isotopically-labelled glycan corresponding to a glycan        associated with a disease or disorder;    -   (iii) adding the tagged standard to a sample that has been        obtained from a patient to obtain a doped sample;    -   (iv) analysing the doped sample using mass spectrometry to        obtain ion peaks;    -   (v) comparing the identity and intensity of the ion peaks        associated with the tagged standard with the additional ion        peaks in the spectrum of the doped sample to identify, and        optionally to quantify, the presence of one or more glycans in        the sample;    -   (vi) using the presence of said one or more glycans to diagnose        the disease or disorder.

Samples obtained from patients may be obtained using methods known inthe art. Suitably, as the glycan(s) in the biological material takenfrom the patient may be conjugated to a protein backbone, the sample maybe obtained by taking biological material from a patient and removingglycan material from the protein backbone enzymatic or chemical(hydrazinolysis) treatment. Suitably, the resultant material may bepurified.

In a further aspect, the present invention provides a kit foridentifying a glycan in a sample, the kit comprising:

-   -   (a) a tagged standard, the tagged standard comprising one or        more isotopically-labelled glycans;    -   (b) instructions for doping a sample suspected of containing a        glycan with the tagged standard to obtain a doped sample and        analysing the doped sample using mass spectrometry.

Optionally, the kit may include mass spectrometry data for the taggedstandard which may facilitate identification of analytes in the samplethrough easy identification of the ion peaks associated with the taggedstandard.

The instructions may further comprise the step of comparing the ionpeaks associated with the tagged standard with the additional ion peaksin the mass spectrum.

In some embodiments, the tagged standard is a mixture ofisotopically-labelled glycans known to be a combination associated witha particular disease, disorder or biological process.

Embodiments of the present invention will now be described by way ofexample and not limitation with reference to the accompanying figures.However various further aspects and embodiments of the present inventionwill be apparent to those skilled in the art in view of the presentdisclosure.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Unless context dictates otherwise, the descriptions and definitions ofthe features set out above are not limited to any particular aspect orembodiment of the invention and apply equally to all aspects andembodiments which are described.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A representative portion of a mass spectrum of a doped sampleshowing the ion peaks associated with an isotopically-labelled glycanand a corresponding analyte. The peaks indicates show the measuredintensity of the ion peaks associated with the isotopically-labelledglycans of the tagged standard and the corresponding analyte glycans ofthe sample.

FIG. 2. A schematic representation of possible combinations of enzymaticelongation steps to afford isotopically-labelled N-glycans.

FIG. 3. The synthesis of isotopically labelled[(2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-[2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-α,β-D-glucopyranose,wherein each acetyl group is ¹³C₂-isotopically-labelled.

FIG. 4. The synthesis of isotopically labelled tri-antennary[(2-acetamido-β-D-glucopyranosyl)-(1→2)-α-D-mannopyrannosyl]-(1→6)-[di-(2-acetamido-β-D-glucopyranosyl)-(1→2)-(1→4)-α-D-mannopyrannosyl]-(1→3)-β-D-mannopyranosyl-(1→4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-1,3,6-tri-O-benzyl-2-deoxy-β-D-glucopyranoside,wherein each acetyl group is ¹³C₂-isotopically-labelled.

FIG. 5. Glycans obtained by chemo-enzymatic synthesis: enzymatictruncation of ¹³C₈-G0.

FIG. 6. Glycans obtained by chemo-enzymatic synthesis: fucosylation of¹³C₆-MGn3.

DETAILED DESCRIPTION Definitions

Isotopically-Labelled

As used herein, isotopic labelling, isotopically-labelled, and othersimilar terms are used as is understood in the art. Specifically, anisotopically-labelled compound is a compound in which at least one atomof known position is enriched with an isotope other than the mostabundant naturally-occurring isotope for that element. For example,methane may be ¹³C-isotopically-labelled, and have the structure ¹³CH₄,or deuterium-labelled. Deuterium-labelled methane may refer to acompound in which one or more of the four hydrogen atom positionsassociated with methane are enriched with ²H (D). Commondeuterium-labelled methane structures include CDH₃ and CD₄.

Isotopic-labelling refers to isotopic enrichment above naturalabundance. Preferably, the isotopic purity at the enriched position isgreater than 50%. For example, in ¹³C-isotopically-labelled methane,this means that 50% or more of the individual molecules comprise a ¹³Catom. In embodiments of the present invention, the isotopic purity atthe enriched position(s) is preferably greater than 80%. Morepreferably, the isotopic purity at the enriched position(s) is greaterthan 90%.

In some preferred embodiments, the isotopic purity at the enrichedposition(s) is greater than 95%.

In some preferred embodiments, the isotopic purity at the enrichedposition(s) is greater than 97%.

In some preferred embodiments, the isotopic purity at the enrichedposition(s) is greater than 98%.

In some preferred embodiments, the isotopic purity at the enrichedposition(s) is greater than 99%.

Acyl Group

As used herein, an acyl group is a functional group derived by theremoval of a hydroxyl group from a carboxylic acid. Common acyl groupsinclude formyl (methanoyl), acetyl (ethanoyl), propionyl (propanoyl),benzoyl, and acrylyl (propenoyl). Other acyl groups of biologicalrelevance include, but are not limited to, hydroxyethanoyl (glycolyl)and acyl groups derived from C₄₋₁₈-fatty acids (for example, butanoyl,hexanoyl, octanoyl, decanoyl, etc.) and hydroxylated fatty acids.

Acylation is the process of adding an acyl group to a compound using anacylating agent. In the context of the present invention, acylationoccurs at a nucleophilic functional group, for example, an amino groupor a hydroxyl group. Where more than one nucleophilic group is present,the order in which groups are acylated is determined by nucleophilicityand steric factors. Common acylating agents include acyl chlorides andacid anhydrides.

Isotopically-labelled acyl groups are those in which at least one atomof known position is enriched with an isotope other than the mostabundant naturally-occurring isotope for that element, as definedherein. Examples include, but are not limited to, all combinations of¹²C, ¹³C, ¹⁴C, ¹⁸O, ¹⁸O, H and D of formyl, acetyl, propionyl, benzoyl,and acrylyl groups available. Other isotopically-labelled acyl groupsmay also be used in methods of the present invention as appropriate. Forexample, an isotopically-labelled hydroxyethanoyl group (such as, forexample, 1-¹³C₁- or ¹³C₂-hydroxylethanolyl) may be used to provide anisotopologue of a glycan containing an N-glycolylneuramic acid unit.Similarly, isotopically-labelled acyl groups derived from fatty acidsmay be used to provide isotopologues of, for example, Lipid A.

In some embodiments, the isotopically-labelled acyl group is anisotopically-labelled acetyl group. Preferred isotopically-labelledacetyl groups Ac* include:

—(¹³C═O)¹³CH₃, —(C═O)¹³CH₃, —(¹³C═O)CH₃,

—(C═O)CD₃, —(¹³C═O)¹³CD₃, —(C═O)¹³CD₃, —(¹³C═O)CD₃,

—(¹⁴C═O)¹⁴CH₃, —(C═O)¹⁴CH₃, —(¹⁴C═O)CH₃,

—(C═¹⁷O)CH₃, or —(C═¹⁸O)CH₃.

In preferred embodiments of the present invention, theisotopically-labelled acetyl group Ac* is selected from: —(¹³C═O)¹³CH₃,—(C═O)¹³CH₃, or —(¹³C═O)CH₃. In particularly preferred embodiments ofthe present invention, the isotopically-labelled acetyl group Ac* is—(¹³C═O)¹³CH₃.

Acylating agent as used herein is used as is understood in the art, thatas, as a chemical reagent that provides an acyl group. Commonly usedacylating agents include acyl chloride and anhydrides of carboxylicacids, although other acylating agents and methods will be apparent toone skilled in the art and may include, for example, the product of areaction between a carboxylic and a suitable coupling reagent. In someembodiments, the isotopically-labelled acylating agent is an acylchloride. Suitable acyl chlorides may be commercially available, or maybe obtained using methods known in the art, for example throughtreatment of the corresponding carboxylic acid with thionyl chloride oroxalyl chloride.

In other embodiments, the isotopically-labelled acylating agent is ananhydride of a carboxylic acid, preferably an anhydride of acetic acid.In some embodiments, the isotopically-labelled acetylating agent isselected from: (¹³CH₃ ¹³C═O)₂, (¹³CH₃C═O)₂, (CH₃ ¹³C═O)₂, (CD₃C═O)₂,(¹³CD₃ ¹³C═O)₂, (¹³CD₃C═O)₂, or (CD₃ ¹³C═O)₂. In some preferredembodiments, the isotopically-labelled acetylating agent is (¹³CH₃¹³C═O)₂.

In some embodiments, a ¹⁴C-acylating agent, preferably a ¹³C-labelledacetic anhydride, may be used. The resultant glycans, labelled with ¹⁴C,may have utility as standards for glycan quantification usingautoradiography.

Protecting Group

As used herein, protecting group refers to a moiety that is introducedinto a molecule by chemical modification of a functional group in orderto obtain chemo-selectivity during a subsequent reaction or to preventunwanted degradation or side-reactions during subsequent reaction. Aprotecting group may also be referred to as a masked or masking group ora blocked or blocking group. By protecting a reactive functional group,reactions involving other unprotected reactive functional groups can beperformed, without affecting the protected group; the protecting groupmay be removed, usually in a subsequent step, without substantiallyaffecting the remainder of the molecule. See, for example, ‘ProtectiveGroups in Organic Synthesis’ (T. Green and P. Wuts, Wiley, 1999).

Examples of protecting groups are well-known in the art, and thefollowing examples are provided for illustration and not by way oflimitation.

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a methoxymethyl(MOM) or methoxyethoxymethyl (MEM) ether; a benzyl (Bn), benzhydryl(diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl ort-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH₃, —OAc) orbenzoyl ester (—OC(═O)Ph, Bz).

For example, an aldehyde or ketone group may be protected as an acetalor ketal, respectively, in which the carbonyl group (>C═O) is convertedto a diether (>C(OR)₂), by reaction with, for example, a primaryalcohol. Thio-acetals and thio-ketals are also known in the art.

For example, a polyhydric moiety may be protected as an acetal group, inwhich for example two hydroxyl groups on carbon atoms adjacent to eachother (HO—CR₂CR₂—OH; often called a glycol group) react with an aldehydeor ketone to from a ring comprising an —O—CR₂—O— linkage, as shownbelow.

Acetals are typically formed under dehydrating conditions (for example,under Dean-Stark conditions or using a Soxhlet extractor) with acidcatalysis and may be removed by acid catalysis and an excess of water,or by other methods known in the art.

For example, an amine group may be protected as an amide or a urethane,for example, as: a methyl amide (—NHCO—CH₃); a benzyloxy amide(—NHCO—OCH₂C₆H₅, —NH-Cbz); as a t-butoxy amide (—NHCO—OC(CH₃)₃,—NH-Boc); a 2-biphenyl-2-propoxy amide (—NHCO—OC(CH₃)₂C₆H₄C₆H₅,—NH-Bpoc), as a 9-fluorenylmethoxy amide (—NH-Fmoc), as a6-nitroveratryloxy amide (—NH-Nvoc), as a 2-trimethylsilylethyloxy amide(—NH-Teoc), as a 2,2,2-trichloroethyloxy amide (—NH-Troc), as anallyloxy amide (—NH-Alloc), as a 2(-phenylsulphonyl)ethyloxy amide(—NH-Psec); or, in suitable cases, as an N-oxide (>NO⁻) or azide.

In some embodiments of the present invention, amine functions inhexosamine sugar donors are protected by phthalimide, TRoc,trichloroacetyl, dimethylacetyl groups. This facilitates β-selectiveformation of glycosidic bonds and prevents unwanted oxazoline formationin these reactions. In some embodiments, amine functions may beprotected as azides, which may facilitate the stereoselective formationof α-glycosidic linkages.

For example, a carboxylic acid group may be protected as an ester, forexample, as: a C₁₋₇ alkyl ester (e.g. a methyl ester; a t-butyl ester);a C₁₋₇ haloalkyl ester (e.g., a C₁₋₇ trihaloalkyl ester); a triC₁₋₇alkylsilyl-C₁-₇ alkyl ester; or a C₅-₂₀ aryl-C₁-₇ alkyl ester (e.g. abenzyl ester; a nitrobenzyl ester); or as an amide, for example, as amethyl amide.

In some embodiments, the present invention uses orthogonal protectinggroup strategies to assemble oligosaccharides andoligosaccharide-containing structures. Orthogonal protection is astrategy known in the art, and involves judicious selection of multipleprotecting groups to enable deprotection of one or more functionalgroups of a molecule using a dedicated set of reaction conditions withaffecting other protecting groups elsewhere in the molecule. Forexample, one protecting group used may be acid labile (e.g. an acetal),another protecting group used may be base labile (e.g. an FMOC group),while a further protecting group used may be removed using hydrogenationconditions (e.g. a benzyl ether). As described herein, where multiplepositions within a structure may each independently be a protectinggroup, said protecting groups may be the same or different. Differentprotecting groups may be orthogonal to each other and consequentlyfacilitate chemo-selective reaction through selective deprotection ofone protecting group in the presence of another. Purely by way ofexample, in an oligosaccharide comprising the motif

wherein each R³ is independently a protecting group, each R³ may be aprotecting group of the same type, or each R³ may be independently thesame or different to any other R³ group. Through use of R³ protectinggroups that are orthogonal, selective deprotection and reaction canoccur at C2, C3, C4 or C6.

Calculation of Concentrations Using Isotopic Dilution

Methods according to the present invention and isotopically-labelledglycans provided by the present invention can be used to determine theconcentration of an analyte of interest, for example, a natural glycan,in a sample. Suitable samples may include glycans released fromproteins, natural glycoconjugates, and the products of recombinantprotein production.

In some methods according to the present invention, a sample suspectedto contain at least one glycan is obtained following, for example,release from a protein by hydrazinolysis or enzymatic cleavage withpeptide glycosidase. To this sample, a known amount of a “taggedstandard” is added to obtain a doped sample. The tagged standardcomprises at least one isotopically-labelled glycan of knownconcentration, and in some embodiments comprises a mixture ofisotopically-labelled glycans with known concentrations of allcomponents.

The doped sample is then analysed using mass spectrometry to acquirespectra. Optionally, during the analysis and acquisition, informationregarding fragmentation of selected ions may be obtained. Thisfragmentation analysis may aid determination of both the overallstructure of the glycan of interest and of the relative and absoluteweaknesses of bonds present. This is of especial relevance to methodsand embodiments of the present invention in which isotopically-labelledmonosaccharide units have been introduced into the isotopically-labelledglycan at one or more pre-determined positions in the oligosaccharidesequence, for example, using chemo-enzymatic methods as describedherein.

Ion peaks in the acquired spectra are then assigned (being identifiableowing to fixed mass increments) and may be quantified through comparisonwith the ion peaks known to be associated with the tagged standard.

For example, a particular natural N-glycan having a[(2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-[2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-α,β-D-glucopyranosemotif may be identified through use of a tagged standard comprising anisotopologue in which the acetyl groups in this heptasaccharide motifare each ¹³C₂-isotopically-labelled, which may be obtained as describedherein. This results in an isotopically-labelled N-glycan having massincremented 8 Da relative to the natural N-glycan, but with thecorresponding associated mass spectrometry ion envelope (as shown inFIG. 1).

Furthermore, the amount of the analyte glycan may be quantified throughcomparison of the ion peak intensities, thereby allowing the amount ofthe analtye glycan to be quantified (the peak intensities of eachisotopologue are proportional to their amounts in the sample). As theisotopically-labelled glycan ionises with the same efficiency as thecorresponding analyte glycan, the relative intensities are proportionalto their relative concentrations (Equation 1). The method further allowsfor the quantification of an analyte in complex mixtures comprisingmultiple glycans, both in terms of amount and relative abundance(Equations 2, 3, and 4). Using methods of the invention and theseequations, analytes in complex mixtures can be quantified. It will beunderstood that use of Equations 1 to 4 is generally applicable tomethods of the present invention, and that Equations 1 to 4 areexplained without limitation with reference to this example method. Forsimplicity, “light” refers to non-isotopically-labelled glycans and“heavy” to the corresponding higher molecular weightisotopically-labelled glycans.

-   I_(i) Peak intensity of the “light” isotopologue i-   I*_(j) Peak intensity of the “heavy” isotopologue j-   m_(i) Amount of the “light” isotopologue i-   m*_(j) Amount of the “heavy” isotopologue j-   m_(T) Total amount of the “light” glycan in the sample-   m*_(T) Total amount of the “heavy” glycan in the sample-   X_(i) Relative abundance of the “light” isotopologue i in the    non-isotopically analyte glycan-   X*_(j) Relative abundance of the “heavy” isotopologue j in the    isotopically labelled glycan

$\begin{matrix}{\frac{I_{i}}{m_{i}} = \frac{I_{j}^{*}}{m_{j}^{*}}} & \left\lbrack {{Equation}\mspace{14mu} 1} \right\rbrack \\{m_{i} = {m_{T} \times X_{i}}} & \left\lbrack {{Equation}\mspace{14mu} 2} \right\rbrack \\{m_{j}^{*} = {m_{T}^{*} \times X_{j}^{*}}} & \left\lbrack {{Equation}\mspace{14mu} 3} \right\rbrack \\{m_{t} = \frac{I_{i} \times m_{T}^{*} \times x_{j}^{*}}{I_{j}^{*} \times X_{i}}} & \left\lbrack {{Equation}\mspace{14mu} 4} \right\rbrack\end{matrix}$

It will be appreciated that while the above equations provide reasonablequantification of the glycans in a sample by comparison to their “heavy”corresponding isotopologue standard, for complex mixtures and spectra inwhich certain peaks are detected at saturated concentration, moredetailed methods may be desirable. For example, if the most abundantpeak of a standard is saturated, simply using that peak in the aboveequations may give an inaccurate quantification. To address thisproblem, the following provides details of isotopic dilution analysis(study of linearity and selection of internal standard isotopologues forthe calculation of the glycan concentration in a sample). This methoduses the ion peaks associated with different “heavy” isotopologues tocalculate a function. This function may be used to relate the peakintensity of the peak to be quantified to the amount of the isotopologueassociated with that peak, thereby mitigating this potential inaccuracy.Once again, the following is provided for illustration and withoutlimitation.

I. Linearity Determination

Knowing the total amount of “heavy” isotopologues for a given glycan(m_(T)*) added to the sample and the corresponding relative abundance(X_(j)*) of each of its constituent “heavy” isotopologues, it ispossible to calculate the amount of each “heavy” isotopologue (m_(j)*)in the sample using equation 3. This may account for different synthetic“heavy” isotopologues, for example, having differently isotopicallylabelled acetyl groups, and/or the various peaks associated with theisotopic envelope for a given “heavy” glycan. Suitably, the variouspeaks associated with the isotopic envelope are used. The theoreticalabundances of these isotopic envelope peaks may be derived from theknown natural abundances of the various isotopes, calculated as aprobability given the empirical formula of the molecule.

By using the ion peak intensities obtained for these different “heavy”isotopologues of a glycan in the isotopically-labelled standard (I_(j)*)and their relative abundances (m_(j)*), a function correlating peakintensities and glycan amount can be calculated by linear regression (Ibeing a function of m):I=bm+a  [Equation 5]:

where the coefficients b and a correspond to the slope and theintercept, respectively, which have been calculated by minimum leastsquares fit (Equations 6 and 7).

$\begin{matrix}{b = \frac{\sum{\left( {m_{j}^{*} - \overset{\_}{m_{j}^{*}}} \right)\left( {I_{j}^{*} - \overset{\_}{I_{j}^{*}}} \right)}}{\sum\left( {m_{j}^{*} - \overset{\_}{m_{j}^{*}}} \right)^{2}}} & \left\lbrack {{Equation}\mspace{14mu} 6} \right\rbrack \\{a = {\overset{\_}{I_{j}^{*}} - {b\;\overset{\_}{m_{j}^{*}}}}} & \left\lbrack {{Equation}\mspace{14mu} 7} \right\rbrack\end{matrix}$

The coefficient of determination R² can be also calculated and used as ameasure of the fitting quality. If R² is lower than a given value, forexample, if R² is less than 0.99, the data point corresponding to themost abundant “heavy” isotopologue may be discarded and the function andR² are calculated again by linear regression. This process may berepeated until all the linearity conditions defined for R² are matched.This iterative process improves the accuracy of the function.

Once a function with the appropriate R² is obtained, the linearity rangedefined is the limits of the maximum and minimum peak intensity and thecorresponding glycan isotopologue amount values. All the peaks withinthis linear range may be considered of sufficient quality to allow avery accurate quantification.

II. Calculation of the Amount of Non-Labelled Glycan in the Sample

As described, a linear function may be obtained using the knownproperties of a tagged standard “heavy” glycan isotopologue mixture.These “heavy” glycans must give peak intensities in the previouslyestablished linear range and with a suitable minimum signal to noiseratio, for example, higher than five.

This way, the amount of an individual “light” glycan isotopologuesatisfying the above criteria (within the maximum and minimum peakintensities) (m_(i)) can be calculated from the function obtained asdescribed above and improved by the by linear regression iterativeimprovement described above.

$\begin{matrix}{m_{i} = \frac{\left( {I_{i} - a} \right)}{b}} & \left\lbrack {{Equation}\mspace{14mu} 8} \right\rbrack\end{matrix}$

Because the “light” glycan itself has an isotopic envelope associatedwith its parent mass spectrometry peak (for example, the naturalabundance of ¹³C) a more accurate analysis of the total amount of theanalyte glycan corrects for this using the theoretical natural abundanceof that “exact mass” isotopologue (X_(i)), which can be easilytheoretically calculated using the statistical probability of theseisotopes being present.

Thus, once the amount of an isotopologue of the “light” (m_(i)) has beencalculated, and knowing the relative abundance of that isotopologue(X_(i)), the analyte glycan in the sample (m_(T)) can be calculated:

$\begin{matrix}{m_{T} = \frac{\left( {I_{i} - a} \right)}{b \times X_{i}}} & \left\lbrack {{Equation}\mspace{14mu} 9} \right\rbrack\end{matrix}$

Accordingly, in some embodiments, methods of quantitatively determiningthe analyte glycan content of a sample may use a tagged standardcomprising a plurality of “heavy” isotopologues of said analyte glycan,the method including the steps of

-   -   (i) correlating the relative intensities of the ion peaks        associated with each “heavy” isotopologue (I_(j)*) with the        known abundance of that glycan in the standard (m_(j)*) to        obtain I_(j) as a linear function of m_(j);    -   (ii) optionally calculating the coefficient of determination R²        for the correlation and discounting the most abundant ion peak        if the R² value is greater than a pre-determined value;    -   (iii) optionally repeating step (ii) one or more times;    -   (iv) using said function to calculate the amount of a “light”        isotopologue of the analyte glycan;    -   (v) optionally using the total amount of the “light”        isotopologue of the analyte glycan to determine the total amount        of analyte glycan present.

Suitably, the R² value may be great than or equal to 0.99.

Furthermore, the present invention further provides a method ofidentifying a “light” isotopologue in a sample, the method comprisingadding a known amount of a tagged standard comprising a plurality ofcorresponding “heavy” isotopologues (said “heavy” isotopologues beingisotopically labelled), analysing the mixture by mass spectrometry andquantifying the amount of “light” isotopologue by comparison of therelative intensity of the ion peaks associated with the “heavy”isotopologues and with the “light” isotopologue.

This quantification may include the steps of:

-   -   (a) correlating the relative intensities of the ion peaks        associated with each “heavy” isotopologue (Ij*) with the known        abundance of that isotopologues in the standard (mj*) to obtain        Ij as a linear function of mj;    -   (b) optionally calculating the coefficient of determination R²        for the correlation and discounting the most abundant ion peak        if the R² value is greater than a pre-determined value;    -   (c) optionally repeating step (ii) one or more times;    -   (d) using said function to calculate the amount of analyte        “light” isotopologue in the analyte sample;    -   (v) optionally using the total amount of the “light”        isotopologue to determine the total amount of analyte glycan        present.

It will be appreciated that the method may be applied to any suitablemolecules available in isotopically-labelled form, the method beingsuitable for the glycan standards described herein but not necessarilylimited to glycan molecules.

Fragmentation

The generation and analysis of molecular fragment ions during massspectrometry experiments is of considerable use in structuraldetermination. Various techniques for the generation and detection ofsuch fragment ions are known in the art and include, but are not limitedto, collision-induced dissociation (CID) and tandem mass spectrometry(variously also called MS/MS and MS2). Analysis and quantification ofthese fragments may aid partial or complete structural determination,and may be especially useful for detecting a given molecule in thepresence of other molecules of the same notional molecular weight. Inthe context of the field of the present invention, fragment analysis mayalso be used to identify weaker bond linkages in analytes and todiscriminate between isobaric structures.

In some embodiments of the present invention, isotopically-labelledmonosaccharide units are incorporated chemo-enzymatically into glycanstructures, for example, using the enzymatic elongation methodsdescribed herein. The use of these glycans for the generation offragmentation patterns is of particular value for discriminating betweenisobaric glycan structures using mass spectrometry techniques.

This can be achieved through the identification and/or assignment ofdiagnostic fragments and/or determination of the weakest linkages in aparticular isomer.

Sugar Abbreviations

As used herein, saccharide abbreviations are used as is commonlyunderstood in the art. The suffix “N” indicates the corresponding aminosugar, while “NAc” indicates the corresponding N-acetyl amino sugar.

Glc—glucose

Gal—galactose

Man—mannose

Rha—rhamnose

Fru—fructose

Fuc—fucose

Mur—muramic acid

Neu—neuraminic acid

Kdo—keto-deoxyoctulosonate

Glycans

The term glycan can be used to refer to any saccharide (mono-, oligo- orpoly-) in free form or forming a carbohydrate portion of aglycoconjugate molecule such as a glycoprotein, proteoglycan orglycolipid. Glycans are important molecules involved in virtually everybiological structure and process. Constituent monosaccharides generate amuch greater combinatorial diversity than nucleic or amino acids, andfurther diversity arises from covalent modification of glycans. Thetotal glycan repertoire (glycome) of a given organism is thus much morecomplex and dynamic than the genome or proteome.

Linkages between monosaccharides can be in α- or β-form, chains can belinear or branched and glycan modifications can include acetylation andsulfation. Glycoproteins carry one or more glycans covalently attachedto a polypeptide via N or O linkages.

O-glycans are linked to hydroxyl groups of serine or threonine residues.N-glycans are sugar chains linked via a side-chain nitrogen (N) to anasparagine residue. They share a common pentasaccharide region of twomannose residues, linked separately by α1-3 and α1-6 linkages to acentral mannose, which in turn is linked by a β1-4 linkage to achitobiose core consisting of two β1-4-linked GlcNAc residues. Based onfurther processing of the pentasaccharide, N-glycans are divided intothree main classes: (i) high-mannose complex (iii) hybrid types.

High-mannose N-glycans have only unsubstituted mannose residues(typically 5-9) attached to the chitobiose core. Hybrid N-glycanscontain both unsubstituted terminal mannose residues and mannoseresidues with a GlcNAc, which initiate “antennae” to which additionalmonosaccharides may be added. Complex N-glycans have GlcNAc residuesadded at both α3 and α6 mannose sites, do not have extra-pentasaccharidemannose residues and are found in bi, tri and tetraantennary forms.

Proteoglycans have one or more glycosaminoglycan (GAG) chains attachedthrough a core region ending with a xylose to the hydroxyl groups of aserine residue. The most important glycolipids are glycosphingolipids,which consist of a glycan usually linked via a glucose or galactose tothe terminal hydroxyl group of a ceramide lipid moiety, which iscomposed of the long chain amino alcohol sphingosine and a fatty acid.

Glycan Binding Proteins

Many of the specific biological roles of glycans are mediated viarecognition by glycan binding proteins (GBPs). GBPs include lectins,glycosaminoglycan binding proteins and glycan-specific antibodies.Lectins often bind to terminal regions of glycan chains throughcarbohydrate recognition domains. Due to low affinity binding,multivalent CRD-glycan interactions are often required for interactionswith biological relevance.

Glycan Processing

Glycans are primarily synthesised by glycosyltransferase enzymes whichassemble monosaccharide moieties into glycan chains.

Glycosyltransferase enzymes have in common the property of being able tocatalyse transfer of a monosaccharide of a simple nucleotide sugar donor(for example, UDP-Gal, GDP-Fuc or CMP-Sia) to an acceptor substrate.

Glycoconjugate biosynthesis is initiated by glycosyltransferase enzymeswhich attach saccharides to a polypeptide side chain or sphingolipidbase. For example, in the case of N-glycans, oligosaccharyltransferasetransfers the glycan Glc3Man9GlcNAc2 to the side chain of asparagine.

The majority of glycosyltransferases elongate glycan chains. Linear orbranched chains are built by sequential glycosylation, often by distinctglycosyltransferases. That is, the product of glycosylation by oneenzyme produces the preferred substrate for another. Examples ofglycosyltransferases include galactose-1-phosphate uridyl-transferase(GalT), N-acetylgalatosaminyl-transferase (GalNAcT), fucosyl transferase(FuT) and sialyltransferase (SialT, which catalyze the addition ofgalactose, N-acetylglucosamine, fucose and sialic acid residues,respectively.

Glycosidases are glycan processing enzymes which remove monosaccharidemoieties to form intermediates which are then acted upon byglycosyltransferases. This type of processing is particularly importantin the biosynthesis of N-glycans; action of glycosidase enzymes on theGlc3Man9GlcNAc2 allows formation of intermediates necessary forprocessing ultimately to high-mannose, complex and hybrid type N-glycansdescribed above.

Chemo-Enzymatic Synthesis of Isotopically-Labelled Glycans

Advances in the exploration of microbial resources and improvedproduction of mammalian enzymes have established the use ofglycosyltransferases as an efficient tool for glycan synthesis (Blixt,2006; Ruiz, 2001; Serna, 2010, Zou, 2011). Using the appropriatesequence of regio- and stereo-specific transferase enzymes and sugardonor building blocks, complex glycan structures can be assembledthrough sequential enzymatic elongation. Similarly, it may be desirableto first truncate a core motif, for example, to facilitate preparationof asymmetric isotopically-labelled glycan standards derived from thebiantennary heptasaccharide 18 ¹³C₈G0(Bn₅). This truncation may beachieved by enzymatic hydrolysis.

Accordingly, methods described herein for the synthesis ofisotopically-labelled glycans for use as mass spectrometry standardsinclude an enzymatic derivatisation step.

In some embodiments, methods for the synthesis of isotopically-labelledglycans include the use of an appropriate hydrolase on anisotopically-labelled oligosaccharide as described herein to truncatethe isotopically-labelled oligosaccharide. In other words, the presentinvention may provide methods for the enzymatic truncation of one ormore sugar units from an isotopically-labelled oligosaccharide coremotif.

The resultant truncated oligosaccharide may then itself undergoenzymatic elongation to incorporate one or more further sugar units. Insome embodiments of the present invention, appropriate transferases incombination with suitable sugar donors are used sequentially in astepwise fashion to assemble isotopically-labelled glycans. Thetransferases may be recombinant glycosyltransferases, transglycosidases,endoglycosidases or mutated glycosidases. The resultant glycans may haveutility in methods of the present invention described herein. In someembodiments, the enzymatic elongation step(s) occurs on anoligosaccharide comprising an isotopically-labelled motif as describedherein, which may variously be termed a core oligosaccharide, a coremotif, and an isotopically labelled starting oligosaccharide. In otherwords, in some methods of the present invention, anisotopically-labelled starting oligosaccharide is chemoselectivelyelongated to incorporate additional sugar units, thereby affordingfurther isotopically-labelled glycan standards for use in massspectrometry.

The sugar donor used in each elongation step may optionally beisotopically-labelled. In some embodiments, only the originalisotopically-labelled motif is isotopically-labelled in the resultantglycan. In other embodiments, at least one isotopically-labelled sugarunit is incorporated during the enzymatic elongation step(s). Asdiscussed above, the incorporation of specific isotopically-labelledsugar units at specific positions has utility in the analysis offragmentation patterns in mass spectrometry.

Alternatively, the enzymatic elongation occurs on a motif that is notisotopically-labelled. Instead, one or more isotopically-labelled sugarunits is incorporated during the enzymatic elongation step(s) to affordan isotopically-labelled glycan which may be used as appropriate in themethods of identifying a glycan in a sample as described herein.

The chemoenzymatic elongation step may be repeated multiple times. Forexample, using monosaccharide sugar donors, 20 cycles of chemo-enzymaticelongation may introduce an additional 20 monosaccharide units. It willbe appreciated that further units may be incorporated at the termini ofantennae, or may be incorporated onto one of the sugar units of the coreoligosaccharide.

In some embodiments, the chemoenzymatic elongation step may utilise asugar donor which is a disaccharide or oligosaccharide and/or which isconjugated to a lipid, peptide or protein.

In some embodiments, the enzymatic derivatisation step may comprise oneor more of an epimerisation step, a transglycosylation step, or apost-translational modification step. These may be in addition toelongation or truncation.

FIG. 2 demonstrates use of the method to assemble a variety of glycansand glycan mixtures. Any sugar unit may be isotopically-labelled. Insome preferred embodiments, each acetyl group in the startingheptasaccharide,[(2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-[2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-α,β-D-glucopyranoseis isotopically-labelled. A chemical synthesis of thisisotopically-labelled starting material is described below. Thisisotopically-labelled starting core oligosaccharide is referred toherein as ¹³C₈-G0.

FIG. 2 shows a series of sequences beginning with this startingheptasaccharide ¹³C₈-G0. Incubation with a recombinant core α1,6fucosyltransferase furnishes core fucosylated structure A, which may befurther galactosylated (panel C) and sialylated (panel D). Directgalactosylation of the starting heptasaccharide with a bovine milkgalactosyltransferase in the presence of UDP-galactose accesses bothmono-galactosylated isomers and the fully galactosylated N-glycan (panelE). Further treatment with a recombinant α2,6 SialylT furnishes compoundpanel F. A bisecting GlcNAc residue may be introduced by virtue of arecombinant GnTIII (compound B). Galactosylation of this product thenleads to the panel G bisecting compounds, and subsequent sialylationaffords compound panel H. α-1,6 fucosylation of bisecting compound Aleads to bisecting and core fucosylated glycan I, which may begalactosyleted towards panel J and finally sialylated to afford compound(panel K).

The synthetically-provided isotopically-labelled core oligosaccharidesmay be protected during the enzymatic derivatisation step, that is, theymay have one or more protecting groups. For example, as describedherein, ¹³C₈-G0 may be obtained via ¹³C₈-benzyl[(2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-[2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside,referred to herein as ¹³C₈-G0(Bn₅). This benzylated heptasaccharide mayitself be used as an isotopically-labelled core motif for the enzymaticderivatisation step(s). As described herein, semi-protected core motifsmay be suitable substrates for chemoselective enzymatic elongation andthe presence of the protecting groups may have particular advantages,for example, by acting as chromophores for peak detection during HPLCanalysis and purification and by aiding separation of differentproducts, for example, isomeric glycans.

It will be appreciated that other non-human e.g. plant or parasitespecific glycans may be assessed in a similar fashion and, by startingwith an isotopically-labelled motif as described above, obtained asisotopically-labelled compounds with a fixed mass increment which iseasily detectable in mass spectrometry experiments. Likewise thechemosynthetic preparation of a larger library N-glycans includinghigher branched complex, hybrid and high mannose type glycans withsystematic variations of the number of antennae, branching pattern andcore modifications may be obtainable starting from a very reduced numberof core structures, which are preferably isotopically-labelled andobtainable using methods according to the present invention. A librarybased on these core structures reflects the structural variation ofN-glycans found in eukaryotic glycoproteins and the most common glycanstructures presented on recombinant glycoproteins.

The following discussion relates to modification of the heptasaccharideN-glycan core referred to herein as ¹³C₈-G0. It is provided forillustration, and is not intended to limit the invention. Other glycancores may equally be envisaged.

The partially deprotected ¹³C-labelled N-glycan ¹³C₈-G0(Bn₅) (13) wasevaluated as precursor for the preparation and isolation of asymmetricN-glycan structures. By taking advantage of the hydrophobicity and UVabsorbance of the penta-benzylated glycans, the present inventors havefound that the presence of these 5 benzyl groups in the core N-glycanstructure facilitates the chromatographic separation of differentglycans and even isomeric structures after a partial enzymaticelongation.

A series of experiments in order to control a partial galactosylation ofthe substrate with bovine milk β-1,4-galactosyltransferase wereperformed, showing not only that the semi-protected N-glycan ¹³C₈-G0(Bn₅was a suitable substrate for the enzyme but also the possibility ofobtaining a mixture of G0-G1-G2 structures, all of them ¹³C-labeled. Theanalysis of this mixture by UPLC-MS, in reverse phase using a C18column, permitted the separation of the different compounds and also thetwo isomers of the mono-galactosylated N-glycan ¹³C₈-G1(Bn₅), and alsoquantify their relative composition by the use of the UV-detector. Theuse of proper conditions during the enzymatic transformation of¹³C₈-G0(Bn₅) yielded the mono-galactosylated biantennary N-glycan¹³C₈-G1(Bn₅) in more than 45% in the form of its two different isomers3-LacNAc and 6-LacNAc.

The purification of both mono-galactosylated isomers separately bysemipreparative-HPLC in miligram scale was achieved as confirmed byMALDI analysis and NMR analysis. The complete deprotection of the coreby hydrogenolysis provided two isomeric ¹³C-labeled standards ¹³C₈-G13and ¹³C₈-G16 for use in N-glycan quantitative analysis. The isomer¹³C₈-G1⁶ could also be enzymaticaly fucosylated for the preparation ofthe standard ¹³C₈-G1⁶F, as confirmed by MALDI-Tof MS.

Another strategy to obtain isomeric asymmetric isotopically-labelledN-glycans for use as mass spectrometry standards consisted of the use aβ-galactosidase from Aspergillus oryzae on the bis-galactosylated¹³C₈-G2(Bn₅) compound (Table 1). A different distribution of glycans wasobserved during this transformation, in which the inventors coulddetermine a different activity of the hydrolase on the two isomericmono-galactosylated structures. This different specificity of the enzymeprovided only one of the mono-galactosylated compound in near 50% yieldand the non-galactosylated compound ¹³C₈-G0(Bn₅).

TABLE 1 BE4-61-G2 Galase Time T (° C.) G2 G1 G1(*) G0 1 5 μg 30 mU 5 h30 14 9 44 33 2 5 μg 15 mU 5 h 30 18 10 46 25 3 5 μg  8 mU 5 h 30 35 1043 12 4 5 μg 15 mU 18 h  30 6 4 38 52

The same strategy was employed for the preparation of a variety of¹³C-labelled sialylated standards from the bis-galactosylated¹³C₈-G2(Bn₅) using a α-2,3-sialyltransferase from Pasteurella multocidaexpressed in E. coli to give the two mono-sialylated and thebis-sialylated glycans derived from the biantennary structure. Thismixture of semi-protected compounds was resolved by UPLC-MS, whichallowed the inventors to determine their relative composition (Table 2).

TABLE 2 CMP- 2,3-SialT G2(Bn₅) NeuNAc (new) Time T(° C.) G2 G2A1 G2A1(*)G2A2 1 10 μg/5 nmol  4 eq  5 mU 30 min 37 16 25 23 36 2 20 μg/10 nmol 4eq 10 mU 30 min 37 22 23 23 32

This reaction was also applied to the mixture of partiallygalactosylated compounds G0/G1/G2 obtained previously. The partialα-2,3-sialylation of this mixture yielded a mixture of 9 structureswhich could be resolved by UPLC-MS, identifying the presence of¹³C₈-G0(Bn₅) and ¹³C₈-G2(Bn₅), both isomers of the mono-galactosylatedcompound ¹³C₈-G1(Bn₅), both isomers of the mono-sialylated compound¹³C₈-G1A1 (Bn₅), both isomers of the mono-sialylated compound¹³C₈-G2A1(Bn₅) and the bis-sialylated biantennary structure¹³C₈-G2A2(Bn₅). This strategy can therefore be used to obtain up to 5new sialylated ¹³C-labeled N-glycans after purification anddeprotection.

The sialylation reaction was then performed in the mg scale in order toobtain the corresponding sialylated standards. The previously preparedsemiprotected ¹³C₈-G1³Bn₅ was sialylated with α-2,3-sialyltransferasefrom P multocida obtaining the sialylated compound ¹³C₈-G1³Bn₅. Thereaction was not complete but the sialylated compound could be isolatedby semipreparative-HPLC. Also, the sialylation of ¹³C₈-G2(Bn₅) in mgscale afforded a mixture of the sialylated standards ¹³C₈-G2A1³(Bn₅),¹³C₈-G2A1⁶(Bn₅) and ¹³C₈-G2A2 (Bn₅) which could be separated bysemipreparative-HPLC.

The sialylation reaction of ¹³C₉-G2(Bn₅) with a humanα-2,6-sialyltransferase expressed in recombinant CHO cells was alsocontrolled in order to obtain the corresponding mono- and bis-sialylatedstructures. The reaction using the partially protected ¹³C₈-G2(Bn₅) assubstrate and only one equivalent of sialic acid donor yielded 26% ofthe mono-sialylated compound. By contrast, the use of an excess of donorgave the bis-sialylated compound as the only product. Both compounds,mono- and bis-sialylated, could be resolved by UPLC-MS. This reactionwas also performed on the G0/G1/G2 mixture obtained previously bypartial galactosylation. Analogous to the previous results with theα-2,3-sialyltransferase, the partial sialylation of the mixturecontaining 3 galactosylated compounds afforded a mixture of 4 new¹³C-labeled 2,6-sialylated N-glycans, which could be resolved byUPLC-MS. The relative composition of the mixture was determinedidentifying the bis-2,6-sialylated biantennary N-glycan ¹³C₈-G2S2(Bn₅),the mono-2,6-sialylated compound ¹³C₈-G1S1(Bn₅) into its two isomericforms separately and the other mono-sialylated compound ¹³C₉-G2S1(Bn₅)(Table 3).

TABLE 3 G2 CMP- (Bn₅) NeuNAc 2,6-SialT time G2S2 G2S1 G2 1 10 μg/ 4 eq 1mU 2 h 98 1 1 5 mmol 2 10 μg/ 1 eq 0.25 mU 30 min — 10 90 5 mmol (0.25nmol/ min) 1 h — 15 85 4 h 2 26 72 24 h 7 40 53

The core oliogsaccharide ¹³C₈-G0(Bn₅) can be also modified for thepreparation of other asymmetric glycan standards derived from thebiantennary structure but with only one terminal GlcNAc. These truncatedmono-antennary structures can be obtained by enzymatic hydrolysis of theterminal glucosamines in ¹³C₈-G0(Bn₅). The benzyl groups present in thestarting molecule again help in the purification of the resultingstructures after the enzymatic hydrolysis. For this purpose, a N-acetylglucosaminidase from Conavalia ensiformis was used over the partiallyprotected substrate ¹³C₈-G0(Bn₅). The optimization of the reactionallowed the inventors to obtain a mixture of the starting material, thetwo isomers of the mono-antennary structure ¹³C₆MGn³(Bn₅) and¹³C₆-MGn⁶(Bn₅) respectively and the product of double hydrolysis¹³C₄-Man3(Bn₅). As the glucosaminidase removes ¹³C-labeled GlcNAcmoieties, the resulting glycans have a different degree of labeling,obtaining the two isomeric mono-antennary structures bearing 6 ¹³C atomsand the trimannose glycan with 4 ¹³C atoms instead of the original 8atoms.

The hydrolysis reaction was scaled up using 3 mg of ¹³C₈-G0(Bn₅). Thismixture could be resolved by semipreparative HPLC and the 3 newcompounds were isolated in mg scale. These compounds were subjected tohydrogenolysis for the removal of the benzyl groups affording thecorresponding ¹³C-labeled glycans ¹³C₆-MGn³, ¹³C₆-MGn⁶ and ¹³C₄-Man3(FIG. 5). Also, the enzymatic fucosylation of ¹³C₆-MGn³ yielded thestandard ¹³C₆-MGn³F quantitatively (FIG. 6).

As described previously, partially benzylated compounds can bederivatised by enzymatic reactions. This partial protection isespecially useful when the corresponding reaction gives more than oneproduct, for example, in a partial galactosylation, since this partialprotection allow the separation of resultant mixtures by HPLC.

The triantennary N-glycan 22 has three different positions which can begalactosylated. A partial galactosylation produces seven newisotopically-labelled glycan standard in a single reaction: the N-glycancompletely galactosylated (G3), three compounds with two galactoseresidues (G2a, G2b, G2c) and three compounds a single galactose residue(G1a, G1b, G1c).

Optional Oxazoline Formation

In some embodiments of the present invention, the method of synthesisfurther comprises the step of oxazoline formation at a free anomericposition of an acetyl-hexosamine unit in an oligosaccharide.

Suitable methods for this synthetic step are known in the art andinclude the use of coupling reagents such as CDI, DCC, EDC, and DMC; orthe use of suitable Lewis acid reagents. Other dehydrating reagents orconditions may also be used, including, but not limited to,chloroformamidium-type reagents and acid combinations.

The resultant isotopically-labelled glycan oxazoline may then be used toprepare an isotopically-labelled glycoconjugate. Suitable protocols areknown in the art (see, for example, Rising, 2008). Preferredglycoconjugates include glycoproteins, glycoforms, glycopeptides,peptidoglycans, glycolipids, glycosides and lipopolysaccharides.

In some preferred embodiments, the method of synthesis involves a glycancomprising the motif[(2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-(2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-α,β-D-glucopyranosein which each acetyl group in the motif is isotopically-labelled. Theglycan may comprise further antennary sugar units. Oxazoline formationat the free anomeric position of this glycan enables the preparation ofglycoconjugate isotopologues having a fixed mass increment of at least 6Da relative to the natural glycoconjugate. In some preferredembodiments, none of the further antennary sugar units areisotopically-labelled and the resultant isotopically-labelledglycoconjugate has a fixed mass increment of 8 Da relative to thenatural glycoconjugate.

Glycan Markers in Diseases and Disorders

The biosynthesis of glycans relies on numerous highly-competitiveprocesses involving glycosyltransferases. As a result, glycosylation ishighly sensitive to the nature of the biochemical environment, andglycosylation and changes in glycosylation have been implicated in manydiseases and disorders. Accordingly, in some aspects, the presentinvention is directed to methods for the convenient identification ofso-called glycan markers (particular glycan structures known to beassociated with a disease or disorder). While in some embodiments thepresent invention provides for the identification and quantification ofa single glycan marker in a complex mixture, in other embodiments anumber of glycan markers associated with one or more diseases ordisorders may be identified and quantified in a single experiment.

In order to assist in the identification of signature combinations ofglycan markers associated with a particular disease or disorder, in somepreferred embodiments the tagged standard is a mixture comprisingisotopically-labelled isotopologues of a combination, and optionally inthe appropriate proportional amounts, known to be associated with adisease or disorder. In this way, pre-mixed tagged standards comprisingone or more isotopically-labelled glycans may be used in methods of theinvention for the determination of the presence of particular glycansignatures, and consequently in methods of diagnosis of diseases anddisorders associated with those signatures.

Diseases and disorders for which suitable tagged standards comprisingone or more isotopically-labelled glycans may be used include:

cancer;

cardiovascular disorders, for example, stroke, myocardial infarction,hypovolemic stroke, atherosclerosis;

inflammatory skin diseases;

diabetes mellitus;

gastrointestinal disorders, including ulcerative colitis;

liver disorders and diseases;

anaemia;

immunological diseases and disorders, for example, Wiskott-Aldrichsyndrome;

autoimmunological diseases;

arthritis, including rheumatoid arthritis;

infectious diseases;

nephropathy;

neurological disorders, including Alzheimer's disease;

pulmonary disorders; and

congenital disorders of glycosylation.

The above list is provided not by way of limitation and it will beunderstood that the methods described herein are of relevance to thedetection, identification, and/or quantification of any glycan biomarkerknown to be associated with a disease or disorder.

It will be appreciated that the present invention provides for manyuseful applications in biopharmaceutical glycol-profiling. The followingillustrative examples are provided to illustrate the variety of uses towhich the isotopologues and methods described herein may be applied:

-   -   Rapid identification of production batches and production sites        via a quantitative singular glycan fingerprint for a given        product. This could help to identify biosimilars packaged as        originals and to track batch original and identity.    -   Precise and quantitative detection of mAb glycans with known        effector functions (influencing the binding of the Fc part to        the Fc receptor) or important effects on the circulatory        half-life. These include glycans with core fucose, terminal        galactose, terminal sialic acid and high mannose glycans (the        latter will be preferentially engage with mannose receptor of        e.g. macrophages leading to the removal of the drug from        circulation).    -   General rapid and quantitative glycan profiling, and        monosaccharide composition, degree of branching, sialylation,        fucose content etc. in high-throughput applications in the        biopharmaceutical industry like clone selection, process        development, batch release through to IND filing.    -   The particular use of fucosylated and sialylated glycans        standards or any other labile glycan as internal standards in        the glycan profiling by MALDI-Tof MS to quantify and monitor        loss or migration of these monosaccharides and to optimize        acquisition parameters to avoid the loss of these residues.    -   The production of kits with the exact glycan composition of an        originator therapeutic mAb or glycoproteins to guide the        biosimilar producer in clone selection and process development.    -   The use of internal standards for the absolute quantification of        glycoforms within mixtures to aid in relating efficacy        experiments to glycosylation, and in the last instance determine        efficacy of a particular glycoform.

EXAMPLES

The following examples are set forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how topractise the invention, and are not intended to limit the scope of theinvention.

Synthesis of an N-Glycan Heptasaccharide Core

The following synthesis is numbered with respect to the correspondingchemicals structures shown in FIG. 3.

Benzyl4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (2)

A solution of benzyl alcohol (54 μL, 0.525 mmol, 1.5 eq) and 1 (250 mg,0.350 mmol, synthesized according to Serna S., Kardak B., Reichardt N.,Martin-Lomas M., Tetrahedron Asymmetry, 2009, 20, 851-856) withmolecular sieves in dry DCM was stirred for 45 min at room temperature.The mixture was cooled to 0° C. and TMSOTf (6 μL, 0.035 mmol, 0.1 eq) isadded. After 1 h, the reaction was quenched with triethylamine, filteredthrough a plug of celite and concentrated. The crude residue waspurified by flash chromatography hexane:EtOAc 9:1 to give the titlecompound (198 mg, 90%).

Rf 0.39 (toluene:EtOAc 9:1); [α]_(D) ²⁰=+9.2 (c=0.5, CHCl₃); ¹HNMR (500MHz, CDCl₃) δ 7.87-7.48 (m, 4H, Phth), 7.40-7.27 (m, 5H, Ph), 7.13-6.96(m, 7H, Ph), 6.95-6.85 (m, 3H, Ph), 5.19-5.09 (m, 2H, H-1, H-4), 4.81(d, J=12.3 Hz, 1H, CH₂ Bn), 4.61-4.54 (m, 3H, CH₂ Bn), 4.50 (d, J=12.4Hz, 1H, CH₂ Bn), 4.42 (dd, J=10.7, 8.9 Hz, 1H, H-3), 4.34-4.27 (m, 2H,H-2, CH₂ Bn), 3.75 (dt, J=9.7, 4.6 Hz, 1H, H-5), 3.68-3.60 (m, 2H, h-6),1.94 (s, 3H, CH₃ Ac); ¹³C NMR (CDCl₃) δ:169.8, 138.1, 137.9, 137.1,133.9, 131.7, 128.5, 128.3, 128.2, 128.0, 127.9, 127.8, 127.8, 127.8,127.5, 123.4, 123.4, 97.3(C-1), 73.9, 73.8, 73.6, 72.6, 71.0, 69.9,55.6, 21.0; HRMS (ESI): m/z: calcd C₃₇H₃₅NO₆Na: 644.2260 (M+Na)⁺, found644.2294.

Benzyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (3)

To a solution of 2 (608 mg, 0.978 mmol) in MeOH:CH₂Cl₂ 2:1 (6 mL) NaOMe0.25 M was added (300 μL, 20%). After stirring for 1 h, acidic ionexchange resin was added until pH 7. The solution was filtered,concentrated and purified by flash chromatography to give the titlecompound (430 mg, 76%).

Rf (hexane:EtOAc); [α]_(D) ²⁰=+9.4 (c=0.5, CHCl₃); ¹H NMR (500 MHz,CDCl₃) δ 7.89-7.50 (m, 4H, Phth), 7.42-7.29 (m, 5H, Ph), 7.13-7.01 (m,7H, Ph), 6.98-6.89 (m, 3H, Ph), 5.20-5.12 (m, 1H, H-1), 4.79 (d, J=12.3Hz, 1H, CH₂ Bn), 4.73 (d, J=12.2 Hz, 1H, CH₂ Bn), 4.67 (d, J=11.9 Hz,1H, CH₂ Bn), 4.61 (d, J=12.0 Hz, 1H, CH₂ Bn), 4.52 (d, J=12.3 Hz, 1H,CH₂ Bn), 4.48 (d, J=12.3 Hz, 1H, CH₂ Bn), 4.29-4.19 (m, 2H, H-2, H-3),3.90-3.78 (m, 3H, H-6, H-6, H-4), 3.65 (dt, J=9.7, 4.9 Hz, 1H, H-5),2.96 (br s, 1H, OH); ¹³C NMR (CDCl₃) δ:168.1, 167.8, 138.3, 137.8,137.2, 133.8, 131.7, 128.6, 128.2, 128.0, 128.0, 127.9, 127.7, 127.7,127.5, 123.4, 123.3, 97.5(C-1), 78.7, 74.4, 73.9, 73.7, 70.9, 70.8,55.5; HRMS (ESI): m/z: calcd C₃₅H₃₃NO₇Na: 602.2155 [M+Na]⁺, found602.2128.

Benzyl2-O-acetyl-4,6-O-benzylidene-3-O-(2-naphthylmethyl)-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside(5)

A solution of 1 (400 mg, 0.69 mmol) and 4 (905 mg, 0.83 mmol, 1.2 eq,synthesized according to Serna S., Kardak B., Reichardt N., Martin-LomasM., Tetrahedron Asymmetry, 2009, 20, 851-856) in dry CH₂Cl₂ with 3Amolecular sieves was stirred for 1 h at room temperature. To thismixture TMSOTf (12 μL, 0.07 mmol, 10%) was added at room temperature andthe reaction stirred until TLC showed complete conversion of thestarting material (1h). The reaction was quenched by addingtriethylamine (20 μL), filtered through a plug of celite andconcentrated. The crude residue was purified by flash chromatography toobtain the title compound (750 mg, 73%).

Rf 0.17 (hexane:EtOAc 3:1); [α]_(D) ²⁰=−4.9 (c=0.5, CHCl₃); ¹H NMR (500MHz, CDCl₃) δ 7.91-7.58 (m, 10H), 7.57-7.28 (m, 12H), 7.23-7.13 (m, 4H),7.12-6.87 (m, 13H), 6.81-6.68 (m, 3H), 5.53 (s, 1H, CHPh), 5.51 (dd,J=3.3, 1.3 Hz, 1H, H-2C), 5.27 (d, J=8.3 Hz, 1H, H-1B), 4.95 (d, J=8.4Hz, 1H, H-1A), 4.88 (d, J=12.1 Hz, 1H, CH₂ Bn), 4.83 (d, J=12.8 Hz, 2H,2×CH₂ Bn), 4.7-4.67 (m, 3H, 2×CH₂ Bn, H-1C), 4.57-4.47 (m, 4H, 2×CH₂Bn), 4.42 (d, J=12.1 Hz, 1H, 1×CH₂ Bn), 4.40-4.35 (m, 2H, 2×CH₂ Bn),4.29 (dd, J=10.7, 8.5 Hz, 1H, H-3B), 4.25-4.08 (m, 6H, H2A, H2B, H-4A,H-4B, H-3A, H-6Ca), 3.90 (t, J=9.6 Hz, 1H, H-4C), 3.68-3.59 (m, 2H,H-6Ba, H-6Bb), 3.59-3.50 (m, 3H, H-6C-b, H-6Aa, H-3C), 3.43 (dd, J=11.1,3.8 Hz, 1H, H-6Ab), 3.30 (ddd, J=9.9, 3.9, 1.7 Hz, 1H, H-5A), 3.23 (dt,J=9.9, 2.2 Hz, 1H, H-5B), 3.13 (td, J=9.7, 4.9 Hz, 1H, H-5C), 2.22 (s,3H, CH₃ Ac); ¹³C NMR (CDCl₃) δ:170.3, 168.6, 167.7, 167.6, 138.7, 138.7,138.5, 137.9, 137.6, 137.3, 135.4, 134.1, 133.9, 133.5, 133.4, 133.1,131.9, 131.8, 131.5, 129.1, 128.6, 128.4, 128.3, 128.2, 128.1, 128.1,127.9, 127.8, 127.7, 127.7, 127.6, 127.6, 127.5, 127.3, 126.9, 126.3,126.1, 126.0, 125.5, 123.8, 123.2, 101.7, 99.4(C-1C), 97.2(C-1A),97.1(C-1B), 79.0, 77.9, 77.0, 76.6, 75.9, 75.8, 74.7, 74.6, 74.4, 74.3,73.2, 72.9, 71.6, 70.6, 69.2, 68.5, 68.3, 67.9, 67.0, 56.6, 55.8, 21.2;HRMS (ESI): m/z: calcd C₈₉H₈₂N₂O₁₉Na: 1506.5443 [M+Na]⁺, found1506.5481.

Benzyl2-O-acetyl-4,6-O-benzylidene-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside(6)

To a solution of 5 (300 mg, 0.202 mmol) in CH₂Cl₂:MeOH 4:1 (1→2 mL), DDQ(138 mg, 0.606 mmol, 3 eq) was added. After 2h, the mixture was dilutedwith EtOAc and washed with saturated NaHCO₃, water and brine. Thesolution was concentrated and purified by flash chromatography to givethe title compound (176 mg, 65%).

Rf 0.37 (hexane:EtOAc 3:2); [α]_(D) ²⁰=−4.6 (c=1, CHCl₃); ¹HNMR (500MHz, CDCl₃) δ 7.94-7.57 (m, 8H, Phth), 7.50-7.27 (m, 15H, Ph), 7.11-6.88(m, 12H, Ph), 6.82-6.68 (m, 3H, Ph), 5.47 (s, 1H, CHPh), 5.31 (dd,J=3.1, 1.2 Hz, 1H, H-2C), 5.26 (d, J=8.2 Hz, 1H, H-1B), 4.95 (d, J=8.4Hz, 1H, H-1A), 4.85 (t, J=12.4 Hz, 2H, CH₂Ph), 4.76 (d, J=1.3 Hz, 1H,H-1C), 4.70 (d, J=12.4 Hz, 1H, CH₂Ph anomeric), 4.62 (d, J=12.0 Hz, 1H,CH₂Ph), 4.50 (d, J=13.3 Hz, 3H, CH₂Ph), 4.47 (d, J=12.0 Hz, 1H, CH₂Ph),4.41 (d, J=12.1 Hz, 1H, CH₂Ph anomeric), 4.37 (d, J=12.4 Hz, 1H, CH₂Ph),4.30-4.23 (m, 1H, H-3B), 4.23-4.08 (m, 6H, H-2B, H-4A, H-2A, H-4B, H-3A,H-6Ca), 3.75-3.67 (m, 2H, H-4C, H-3C), 3.63 (dd, J=7.2, 2.3 Hz, 2H,H-6Ba, H-ABb), 3.59-3.50 (m, 2H, H-6Cb, H-6Aa), 3.43 (dd, J=11.1, 3.8Hz, 1H, H-6Ab), 3.33-3.27 (m, 1H, H-5A), 3.23-3.18 (m, 1H, H-5B), 3.15(dd, J=15.0, 8.2 Hz, 1H, H-5C), 2.20 (s, 3H, CH₃ Ac); ¹³C NMR (CDCl₃)δ:170.6, 168.5, 167.7, 138.7, 138.4, 138.0, 137.3, 137.1, 134.1, 133.9,133.5, 131.8, 131.5, 129.3, 128.6, 128.4, 128.4, 128.1, 128.1, 127.9,127.9, 127.7, 127.6, 127.5, 127.3, 126.9, 126.3, 123.7, 123.2, 102.1,99.3(C-1C), 97.2(C-1A), 97.0(C-1B), 79.1, 78.6, 76.6, 75.7, 74.6, 74.6,74.4, 74.4, 73.3, 72.9, 71.4, 70.5, 69.9, 68.5, 68.3, 67.8, 66.7, 56.6,55.8, 21.1; HRMS (ESI): m/z: calcd C₇₈H₇₄N₂O₁₉Na: 1365.4784 [M+Na]⁺,found 1365.4840.

Benzyl(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside(8)

A solution of 6 (100 mg, 0.074 mmol) and 7 (80 mg, 0.089 mmol, 1.2 eq,synthesized according to Unverzagt, C.; Eller, S.; Mezzato, S.;Schuberth, R. Chem. Eur. J. 2007, 14, 1304-1311) in dry CH₂Cl₂ withmolecular sieves was stirred at room temperature for 1 h. To thismixture, TMSOTf (1.6 μL, 0.007 mmol, 10%) was added and stirred untilTLC showed complete conversion of the starting material (1h). Thereaction was quenched by the addition triethylamine (20 μL), filteredthrough a plug of celite and the filtrate concentrated. The cruderesidue was purified by flash chromatography to give the title compound(116 mg, 76%).

Rf 0.13 (hexane:EtOAc 1:1); [α]_(D) ²⁰=−15.8 (c=0.5, CHCl₃); ¹ NMR (500MHz, CDCl₃) δ 7.95-7.51 (m, 16H), 7.51-7.27 (m, 11H), 7.08-6.89 (m,12H), 6.84-6.68 (m, 3H), 5.48-5.40 (m, 2H, H-3E, CHPh), 5.23 (d, J=7.9Hz, 1H, H-1B), 5.16 (d, J=4.0 Hz, 1H, H-2C), 5.02 (t, J=10.2 Hz, 1H,H-4D), 4.99-4.89 (m, 3H, H-4D, H-1A, H-1D), 4.89-4.77 (m, 4H, H-1E,2×CH₂Ph, H-3D), 4.68 (d, J=12.2 Hz, 2H, CH₂Ph anomeric, CH₂Ph),4.54-4.46 (m, 4H, 3×CH₂Ph, H-1C), 4.41-4.30 (m, 3H, CH₂Ph anomeric,2×CH₂Ph), 4.28-4.05 (m, 8H, H-2E, H-3B, H-4A, H-2B, H-2A, H-3A, H-4B,H-6Ca), 4.00 (dd, J=3.0, 1.7 Hz, 1H, H-2D), 3.93 (dd, J=12.3, 3.3 Hz,1H, H-6Ea), 3.83 (dt, J=9.8, 3.7 Hz, 1H, H-5D), 3.73 (t, J=9.6 Hz, 1H,H-4C), 3.70-3.62 (m, 4H, H-6Eb, H-6 Da, H-6Db, H-6Ba), 3.60-3.51 (m, 3H,H-6Bb, H-6Aa, H-3C), 3.48 (t, J=10.3 Hz, 1H, H-6Cb), 3.38 (dd, J=11.1,3.6 Hz, 1H, H-6Ab), 3.29 (dd, J=9.8, 3.1 Hz, 1H, H-5A), 3.15 (dd, J=9.9,2.1 Hz, 1H, H-5B), 3.00 (td, J=9.7, 5.0 Hz, 1H, H-5C), 2.15 (s, 4H, CH₃Ac, H-5E), 2.05 (d, J=5.8 Hz, 6H, CH₃ Ac), 1.99 (d, J=11.5 Hz, 5H, CH₃Ac), 1.86 (d, J=4.8 Hz, 6H, CH₃ Ac); ¹³C NMR (CDCl₃) δ:170.6, 170.6,170.5, 170.2, 170.1, 169.5, 169.2, 167.7, 167.6, 138.8, 138.7, 138.5,137.9, 137.4, 137.3, 134.3, 134.1, 133.9, 133.5, 131.8, 131.8, 131.5,130.2, 129.0, 128.8, 128.6, 128.3, 128.1, 128.1, 127.9, 127.7, 127.6,127.6, 127.6, 127.3, 127.0, 126.9, 123.8, 123.7, 123.2, 102.4, 98.5(C-1C), 98.0 (C-1D), 97.2 (C-1A, C-1B), 95.8 (C-1E), 78.8, 78.3, 76.7,76.5, 75.9, 75.3, 74.6, 74.5, 74.4, 74.3, 73.5, 72.9, 71.2, 70.6, 70.6,70.5, 69.4, 68.9, 68.6, 68.5, 68.3, 67.5, 66.2, 65.5, 62.9, 61.1, 56.6,55.8, 54.1, 20.9, 20.7, 20.6, 20.6; HRMS (ESI): m/z: calcdC₁₁₀H₁₀₉N₃O₃₆Na: 2070.6689 [M+Na]⁺, found 2070.6689.

Benzyl(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→3)2-O-acetyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside(9)

To a solution of 8 (100 mg, 0.046 mmol) in CH₂Cl₂ (1 mL) at 0° C.,ethanethiol (17 μL, 0.244 mmol, 5 eq) and borontrifluoridediethyletherate (1 μL, 20%) were added. After 2h, triethylamine isadded. The mixture was concentrated and purified by flash chromatography(hexane:EtOAc, 3:1) to give the title compound (75 mg, 83%).

Rf 0.1 (hexane:EtOAc 1:2); [α]_(D) ²⁰=−3.9 (c=0.5, CHCl₃); NMR (500 MHz,CDCl₃) δ 7.94-7.41 (m, 12H, Phth), 7.35-7.22 (m, 10H, Phth, Ph), 7.16(m, 1H, Ph), 7.07-6.93 (m, 12H, Ph), 6.74 (m, 3H, Ph), 5.72 (dd, J=10.7,9.1 Hz, 1H, H-3E), 5.36 (d, J=8.5 Hz, 1H, H-1E), 5.25 (d, J=8.1 Hz, 1H,H-1B), 5.18-5.10 (m, 3H, H-4D, H-4E, H-2C), 4.96-4.91 (m, 2H, H-1D,H-1A), 4.91-4.81 (m, 3H, 2×CH₂ Bn, H-3D), 4.68 (d, J=12.4 Hz, 1H,CH₂Ph), 4.60 (d, J=12.1 Hz, 1H, CH₂Ph), 4.54 (s, 1H, H-1D), 4.53-4.46(m, 3H, 3×CH₂ Bn), 4.43-4.34 (m, 4H, 3×CH₂ Bn, H-2E), 4.29 (dd, J=12.3,4.8 Hz, 1H, H-6E), 4.27-4.13 (m, 5H, H-2B, H-3B, H-4A, H-2D, H-2A),4.13-4.06 (m, 3H, H-6E, H-3A, H-4B), 3.85-3.77 (m, 4H, H-5E, H-5D, H-6Da, H-6Db), 3.75 (t, J=9.5 Hz, 1H, H-4C), 3.68 (dd, J=11.8, 3.4 Hz, 1H,H-6Ca), 3.62 (dd, J=11.6, 1.7 Hz, 1H, H-6Ba), 3.57-3.50 (m, 3H, H-6Aa,H-6Bb, H-6Cb), 3.42 (dd, J=11.1, 3.8 Hz, 1H, H-6Ab), 3.34 (dd, J=9.4,3.5 Hz, 1H, H-3C), 3.31-3.26 (m, 1H, H-5A), 3.22-3.16 (m, 1H, H-5B),2.98 (dt, J=8.9, 4.1 Hz, 1H, H-5C), 2.11 (2×s, J=1.3 Hz, 6H, 2×CH₃ Ac),2.06-2.00 (3×s, 9H, 3×CH₃ Ac), 1.98 (s, 3H, CH₃ Ac), 1.86 (s, 3H, CH₃Ac); ¹³C NMR (CDCl₃) δ:170.9, 170.8, 170.7, 170.3, 170.2, 169.5, 169.5,168.6, 167.7, 138.7, 138.5, 138.4, 137.8, 137.3, 134.5, 133.5, 131.8,131.4, 128.7, 128.4, 128.3, 128.2, 128.2, 128.1, 127.9, 127.6, 127.6,127.5, 127.4, 126.9, 123.8, 123.7, 123.3, 123.2, 98.4(C-1D), 97.7(C-1C),97.2(C-1E), 97.2(C-1A), 97.1(C-1B), 77.6, 77.5, 76.7, 75.4, 74.6, 74.6,74.5, 74.5, 74.4, 73.4, 72.9, 72.0, 70.7, 70.6, 70.5, 69.9, 69.1, 69.0,68.5, 68.2, 65.5, 62.5, 62.1, 62.1, 56.5, 55.8, 54.4, 21.0, 20.9, 20.8,20.7, 20.7, 20.5; HRMS (ESI): m/z: calcd C₁₀₃H₁₀₅N₃O₃₆Na: 1982.6376[M+Na]⁺, found 1982.6331.

Benzyl[(3,4,6-tri-O-acetyl-2-deoxy-2phthalimido-β-D-glucopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→6)]-[(3,4,6-tri-O-acetyl-2-deoxy-2phthalimido-β-D-glucopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→3)]-2-O-acetyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside(11)

A solution of 9 (45 mg, 0.023 mmol) and 10 (30 mg, 0.034 mmol, 1.2 eq,synthesized according to Unverzagt, C.; Eller, S.; Mezzato, S.;Schuberth, R. Chem. Eur. J. 2007, 14, 1304-1311) in dry CH₂Cl₂ (6 mL)with molecular sieves was stirred at room temperature for 1 h. Thismixture was cooled to −40° C., TMSOTf (1 μL, 0.007 mmol, 25%) was addedand the reaction stirred at this temperature until TLC showed completeconversion of the starting material (1h). The reaction was quenched byadding triethylamine (5 μL), filtered through a plug of celite andconcentrated. The crude residue was purified by flash chromatography andpreparative plate gave the title compound (45 mg, 74%).

Rf 0.28 (hexane:acetone 1:1); [α]_(D) ²⁰=−2.8 (c=0.5, CHCl₃); ¹H NMR(500 MHz, CDCl₃) δ 7.88-7.55 (m, 15H, Phth), 7.40 (m, J=7.1 Hz, 1HPhth), 7.33-7.20 (m, 9H, Ph), 7.14 (m, J=5.3, 2.8 Hz, 1H, Ph), 7.06-6.97(m, 3H, Ph), 6.98-6.88 (m, 6H, Ph), 6.84 (m, J=7.3 Hz, 2H, Ph),6.81-6.67 (m, 4H, Ph), 5.69 (dd, J=10.8, 9.1 Hz, 1H, H-1E), 5.61 (dd,J=10.8, 9.2 Hz, 1H, H-1E′), 5.40 (d, J=8.5 Hz, 1H, H-1E), 5.22-5.14 (m,4H, H-4D, H-4E, H-1B, H-1E′), 5.14-5.05 (m, 3H, H-4D′, H-2C, H-4E′),4.94 (dd, J=10.2, 3.4 Hz, 1H, H-3D′), 4.90-4.86 (m, 2H, H-1D, H-1A),4.83 (dd, J=10.2, 3.2 Hz, 1H, H-3D), 4.78 (d, J=12.9 Hz, 1H, CH₂ Bn),4.72 (d, J=12.7 Hz, 1H, CH₂ Bn), 4.68-4.60 (m, 2H, CH₂ Bn), 4.53 (s, 1H,H-1C), 4.52-4.36 (m, 7H, 5×CH₂ Bn, H-2E, H-1D′), 4.36-4.25 (m, 4H,H-2E′,H-6aE CH₂ Bn anomeric, H-4D), 4.23-4.14 (m, 3H, H-3B, H-4A,H-6aE′), 4.14-4.00 (m, 6H, H-2A, H-2B, H-2D′, H-3A, H-4B, H-6bE),3.90-3.83 (m, 2H, H-5E, H-6aD), 3.84-3.70 (m, 7H, H-6bE′, H-4C, H-5D,H-6bD, H-6aD′, H-6bD′, H-6aC), 3.67 (d, J=9.9 Hz, 1H, H-5D), 3.62-3.45(m, 3H, H-6aB, H-6bB, H-6aA), 3.36-3.27 (m, 4H, H-6BA, H-6BC, H-3C,H-5E′), 3.24 (d, J=8.0 Hz, 1H, H-5A), 3.15 (d, J 9.3 Hz, 1H, H-5B), 3.10(dt, J=8.4, 3.9 Hz, 1H, H-5C), 2.13 (s, 3H, CH₃ Ac), 2.09 (s, 3H, CH₃Ac), 2.02 (3×s, 9H, 3×CH₃ Ac), 2.01-1.97 (m, 15H, 5×CH₃ Ac), 1.93 (s,3H, CH₃ Ac), 1.85 (d, J=2.3 Hz, 6H, CH₃ Ac); ¹³C NMR (CDCl₃) δ:171.0,170.9, 170.8, 170.8, 170.7, 170.4, 170.3, 170.2, 169.5, 169.4, 168.3,167.7, 167.5, 138.8, 138.7, 138.4, 138.0, 137.2, 134.5, 134.1, 133.8,133.5, 131.8, 131.7, 131.5, 131.4, 128.7, 128.3, 128.2, 128.1, 128.1,128.0, 127.9, 127.6, 127.5, 127.3, 126.9, 123.7, 123.7, 123.6, 123.2,99.0(C-1D), 98.1(C-1C), 97.8(C-1D′), 97.2(C-1E), 97.2, 97.1, 97.0,(C-1A, C-1B, C-1E′) 78.2, 78.1, 76.7, 75.8, 74.5, 74.4, 74.4, 74.3,73.3, 73.2, 72.8, 71.8, 71.7, 70.7, 70.6, 70.5, 70.4, 70.0, 69.3, 69.1,68.9, 68.5, 68.1, 68.1, 67.3, 65.7, 65.4, 62.5, 62.4, 61.8, 61.6, 56.5,55.7, 54.5, 20.9, 20.9, 20.8, 20.7, 20.7, 20.5; HRMS (ESI): m/z: calcdC₁₃₅H₁₄₀N₄O₅₃Na: 2687.8275 [M+Na]⁺, found 2687.8379.

Benzyl[(2-amino-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-(2-amino-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3))-β-D-mannopyranosyl-(1→4)-2-amino-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1→4)-2-amino-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside(12)

To a solution of heptasaccharide 11 (32 mg, 12 μmol) in MeOH:CH₂Cl₂ 2:1,NaOMe (30 μL, 0.5M, 1.25 eq) was added. After stirring 1h at roomtemperature, MeOH (400 μL) and ethylenediamine (300 μL) were added andthe mixture heated for 3 cycles of 30 min at 120° C. in a microwave. Themixture was concentrated to dryness using toluene and ethanol. The cruderesidue was purified by Sephadex LH-2 column (MeOH:DCM 2:1) to give thetitled compound (17 mg, 83%).

¹H NMR (500 MHz, MeOD) δ 7.52 (d, J=7.1 Hz, 2H, Ph), 7.46-7.25 (m, 21,Ph), 7.21 (dd, J=4.7, 2.0 Hz, 2H, Ph), 5.22 (dd, J=6.8, 4.8 Hz, 2H,CH₂Bn, H-1 Man), 5.17 (d, J=11.5 Hz, 1H, CH₂Bn), 4.98 (d, J=1.9 Hz, 1H,H-1 Man), 4.70-4.56 (m, 5H, 4 CH₂Bn, H-1 Man), 4.50 (s, 2H, CH₂Bn), 4.47(d, J=8.2 Hz, 1H, H-1 GlcN), 4.43 (d, J=7.8 Hz, 1H, H-1 GlcN), 4.39 (d,J=7.9 Hz, 1H, H-1 GlcN), 4.30 (d, J=8.1 Hz, 1H, H-1 GlcN), 4.25-4.12 (m,3H), 4.08 (t, J=9.2 Hz, 1H, H-4 GlcN), 4.02-3.96 (m, 1H, H-6 Glc),3.96-3.76 (m, 12H), 3.76-3.61 (m, 10H), 3.58 (m, 2H), 3.52 (m, 1H),3.48-3.42 (m, 1H), 3.41-3.32 (m, 5H), 3.31-3.19 (m, 4H), 2.83 (td,J=10.7, 8.1 Hz, 2H, H-2 GlcN), 2.77 (d, J=8.4 Hz, 1H, H-2, GlcN), 2.67(dd, J=9.7, 8.0 Hz, 1H, H-2 GlcN); ¹³C NMR from HSQC experiment (126MHz, MeOD) δ 128.48, 127.90, 127.93, 127.38, 127.89, 100.48, 74.34,74.41, 97.67, 70.66, 74.24, 72.70, 99.85, 70.73, 74.33, 72.71, 72.90,101.50, 101.71, 101.96, 101.48, 77.11, 74.70, 70.40, 76.35, 77.63,70.20, 66.17, 61.22, 73.63, 70.36, 66.15, 68.17, 61.02, 68.29, 66.25,60.84, 67.52, 72.89, 68.48, 75.01, 82.05, 82.63, 82.33, 76.99, 70.04,75.54, 75.27, 76.85, 56.82, 56.18, 56.16, 56.22.

Benzyl[(2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-[2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside(¹³C₈) 13 (¹³C₈-G0(Bn₅))

To a solution of heptasaccharide 12 (100 mg, 62.5 μmol) in dry MeOH (2mL) at 0° C., acetic anhydride ¹³C₄ (42 μL, 444 μmol) and NaOMe 0.5M(0.8 mL) were added. After 2 h at 0° C. the mixture was concentrated andpurified by HPLC Sepadex LH-20 (MeOH) to give the titled compound (80mg, 72%). ¹H NMR (500 MHz, MeOD) δ 7.40-7.21 (m, 22H, arom), 7.21-7.14(m, 3H, arom), 5.07 (d, J=1.9 Hz, 1H, H-1D), 4.99 (dd, J=16.3, 12.0 Hz,2H, 2 CH₂Bn), 4.82 (d, J=12.5 Hz, 1H, CH₂Bn), 4.79 (d, J=1.8 Hz, 1H,H-1D′), 4.75 (d, J=12.1 Hz, 1H, CH₂Bn), 4.70-4.66 (m, 2H, H-1C, H-1B),4.66-4.54 (m, 4H, CH₂Bn), 4.46 (dd, J=13.9, 8.2 Hz, 4H, H-1A, H-1E, 2CH₂Bn), 4.31 (d, J=8.4 Hz, 1H, H-1E), 4.11 (d, J=3.1 Hz, 1H, H-2C), 4.08(dd, J=3.4, 1.8 Hz, 1H, H-2D), 3.99 (t, J=8.5 Hz, 2H, H-4A, H-4B),3.96-3.86 (m, 3H, H-2A, H-6Ca, H-6 Da), 3.86-3.75 (m, 9H, H-6Ea, H-2B,H-4C, H-6Aa, H-6Ab, H-3D, H-3D′, H-2D′, H-5D), 3.75-3.53 (m, 14H,H-6E′a, H-6E′b, H-6Eb, H-6D′a, H-6Ba, H-3B, H-2E′, H-2E, H-6Db, H-6D′b,H-6Cb, H-6Bb, H-5D′, H-3A), 3.53-3.41 (m, 6H, H-4D′, H-4D, H-5A, H-3E,H-3E′, H-3C), 3.37-3.24 (m, 4H, H-4E′, H-4E, H-5B, H-5E), 3.20-3.13 (m,2H, H-3E′, H-5C), 2.11 (t, J=5.7 Hz, 3H, Ac), 1.95 (dd, J=18.2, 6.1 Hz,3H, Ac), 1.88-1.83 (m, 3H, Ac), 1.70 (dd, J=18.1, 5.9 Hz, 3H, Ac); ¹³CNMR from HSQC experiment (126 MHz, MeOD): 127.9, 127.8, 127.7, 127.4,100.5(C-1E′), 100.1(C-1A, C-1B, C-1C, C-1E), 99.6(C-1D), 97.2(C-1D′),81.5, 80.8, 80.4, 77.6, 77.1, 76.6, 76.1, 75.9, 74.9, 74.9, 74.0, 73.9,73.8, 73.5, 73.1, 73.0, 72.9, 70.5, 70.3, 70.3, 70.2, 70.2, 68.5, 68.4,67.8, 65.8, 61.9, 61.7, 61.5, 61.0, 55.8, 55.8, 54.5, 22.1, 22.1, 21.6.

[(2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-[2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-α,β-D-glucopyranose(¹³C₈) (14) (¹³C₈-G0(Bn₅))

Heptasaccharide 13 (13 mg, 7.3 μmol) was dissolved in 1 mL of MeOH andpassed through the hydrogenator using a 10% Pd/C cartridge and MeOH assolvent with a flow rate of 1 mL/min, at 50° C. using full hydrogenconditions. The resulting mixture was concentrated, redissolved in waterand purified on a graphite cartridge to give the title compound (7 mg,72%).

¹H NMR (500 MHz, D₂O) δ 5.20 (d, J=2.5 Hz, 0.6H, H-1A_(α-GlcNAc)), 5.13(d, J=1.8 Hz, 1H, H-1D_(α-1,3-Man)), 4.93 (d, J=1.8 Hz, 1H,H-1D′_(α-1,6-Man)), 4.71 (d, J=8.0 Hz, 0.4H, H-1A_(β-GlcNAc)), 4.62 (dd,J=7.8, 4.4 Hz, 1H, H-1B_(β-GlcNAc)), 4.57 (d, J=8.4 Hz, 2H,H-1E_(β-GlcNAc), H-1E′_(β-GlcNAc)), 4.26 (d, J=2.5 Hz, 1H, H-2C), 4.20(dd, J=3.4, 1.6 Hz, 1H, H-2D), 4.12 (dd, J=3.4, 1.7 Hz, 1H, H-2D′),4.04-3.85 (m, 10H), 3.85-3.39 (m, 30H), 2.25-2.13 (m, 6H, 2Ac),1.98-1.88 (m, 6H, 2Ac). ¹³C NMR (126 MHz, D₂O) δ 101.4(C-1B_(β-GlcNAc)), 100.4 (C-1C_(β-1,4-Man)), 99.6 (C-1D_(α-1,3-Man),C-1E_(β-GlcNAc), C-1E′_(β-GlcNAc)), 97.0(C-1D′_(α-1,6-Man)),94.8(C-1A_(β-GlcNAc)), 90.4(C-1A_(α-GlcNAc)), 80.4, 79.6, 79.5, 79.2,76.4, 76.3, 75.8, 75.8, 74.6, 74.4, 74.3, 73.5, 73.4, 73.3, 72.8, 72.5,72.0, 70.2, 70.0, 69.9, 69.6, 69.4, 69.4, 69.2, 67.3, 67.3, 65.8, 65.7,61.7, 61.6, 60.6, 60.1, 60.0, 59.9, 56.1, 55.3, 54.9, 53.6; HRMS (ESI):m/z calculated for C₄₂ ¹³C₈H₈₄N₄O₃₆Na: 1347.5031 [M+Na]⁺, found1347.5131.

Synthesis of a Triantennary Complex N-Glycan Core

The following synthesis is numbered with respect to the correspondingchemicals structures shown in FIG. 4.

Benzyl[di-(O-3,4,6-tri-O-acetyl-2-deoxy-2-phtalimido-β-D-glucopyranosyl)-(1→2)-(1→4)-3,6-di-O-acetyl-α-D-mannopyrannosyl]-(1→3)-2-O-acetyl-4,6-O-benzylidene-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phtalimido-β-D-glucopiranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phtalimido-β-D-glucopyranoside(18)

A solution of acceptor 7 (0.18 g, 0.13 mmol) and donor 17 (0.21 g, 0.16mmol, 1.2 eq) in dry DCM (2 mL), with molecular sieves was stirred atroom temperature for 1 h. TMSOTf (2 μL, 13 μmol, 10%) was added and thereaction was stirred for 1h at room temperature. The reaction wasquenched by the addition of Et₃N (25 μL), filtered through a plug ofcelite and the filtrate concentrated. The crude residue was purified byflash column chromatography (Hexane:EtOAc 2:3), to give the titledcompound (0.27 g, 82%). [α]²⁰ _(D): −24.0 (c 1.05, CH₃C₁). ¹H NMR (500MHz, CDCl₃): 7.90-7.65 (m, 17H, H-arom), 7.54 (m, 2H, H-arom), 7.42-7.26(m, 13H, H-arom), 7.06-6.96 (m, 11H, H-arom), 6.79 (m, 3H, H-arom), 5.76(dd, J=9.0, 10.7 Hz, 1H, H-3E′), 5.44 (m, 3H, H-1E′, H-3E, CHPh), 5.27(d, J=7.8 Hz, 1H, H-1A), 5.18 (m, 2H, H-2C, H-4E′), 4.96 (d, J 8.5 Hz,1H, H-1B), 4.83 (m, 6H, H-4E, 2×CH₂Bn, H-3D, H-1D, H-1E), 4.71, 4.65 (d,J=12.0 Hz, 1H, CH₂ Bn), 4.52 (m, 4H, H-1C, 3×CH₂Bn), 4.39 (m, 3H,3×CH₂Bn), 4.29-4.07 (m, 13H, H-2E′, 2×H-6E′, H-2A, H-2B, H-3A, H-3B,H-4A, H-4B, H-6Ca, H-5E′, H-2E, H-6 Da), 3.98 (m, 3H, H-5D, H-6Ea,H-2D), 3.82 (t, J=10.3 Hz, 1H, H-4D), 3.73 (t, J=8.5 Hz, 1H, H-4C),3.75-3.39 (m, 8H, 2×H-6A, H-6Eb, 2×H-6B, H-3C, H-6Cb, H-6Db), 3.32 (m,1H, H-5B), 3.19 (m, 1H, H-5A), 2.99 (m, 1H, H-5C), 2.26, 2.12 (s, 3H,CH₃ Ac), 2.08 (m, 1H, H-5E), 2.04, 2.03, 2.02, 1.84, 1.83, 1.72, 1.54(s, 3H, CH₃ Ac). ¹³C NMR (126 MHz, CDCl₃): 170.8, 170.5, 170.4, 170.2,170.1, 170.0, 169.5, 169.2, 167.3 138.7, 138.6, 138.4, 137.8, 137.3,137.2, 134.3, 134.0, 133.5, 131.7, 131.4, 128.9, 128.7, 128.5, 128.2,128.1, 127.0, 127.7, 127.6, 127.5, 127.4, 127.2, 126.9, 126.8, 123.8,123.4, 102.3, 98.1 (C-1C), 97.6 (C-1D), 97.1 (C-1A, C-1B), 95.8 (C-1E),94.8 (C-1E′), 78.8, 77.8, 76.6, 75.9, 75.1, 74.5, 74.3, 74.2, 73.3,72.8, 72.7, 71.8, 71.0, 70.8, 70.5, 70.4, 68.6, 68.5, 68.4, 68.1, 67.4,66.0, 63.6, 61.6, 61.1, 56.5, 55.7, 54.8, 54.0, 20.9, 20.7, 20.6, 20.4,20.2.

Benzyl[di-(O-3,4,6-tri-O-acetyl-2-deoxy-2-phtalimido-β-D-glucopyranosyl)-(1→2)-(1-4)-3,6-di-O-acetyl-α-D-mannopyrannosyl]-(1→3)-2-O-acetyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phtalimido-β-D-glucopiranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phtalimido-β-D-glucopyranoside(19)

EtSH (31 μL, 0.25 mmol, 5 eq) and BF₃.OEt₂ (2 μL, 10 μmol, 20%) wereadded at 0° C. to a solution of hexasaccharide 18 (0.14 g, 0.05 mmol) inDCM (2 mL). The reaction mixture was stirred at room temperature untilcomplete conversion (2 hour). Then, it was quenched with Et₃N,concentrated and purified by flash column chromatography (Hexane:EtOAc1:3), obtaining the titled compound (0.12 g, 88%).

[α]²⁰ _(D): +1.9 (c 0.93, CDCl₃). ¹H NMR (500 MHz, CDCl₃): 7.90-7.66 (m,15H, H-arom), 7.32-7.24 (m, 12H, H-arom), 7.17 (m, 1H, H-arom),7.02-6.98 (m, 10H, H-arom), 6.76 (m, 3H, H-arom), 5.78 (dd, J=9.1, 10.7Hz, 1H, H-3E), 5.73 (m, dd, J=9.1, 10.8 Hz, 1H, H-3E′), 5.48 (d, J=7.8Hz, 1H, H-1E′), 5.27 (m, 2H, H-1E, H-1A), 5.22 (t, J=9.8 Hz, 1H, H-4E′),5.15 (d, J=3.4 Hz, 1H, H-2C), 5.10 (t, J=9.7 Hz, 1H, H-4E), 5.05 (dd,J=3.0, 8.5 Hz, 1H, H-3D), 4.96 (d, 1H, J=8.2 Hz, H-1B), 4.86 (m, 3H,CH₂Bn, H-1D), 4.70 (d, J=12.0 Hz, 1H, 1×CH₂Bn), 4.58 (d, J=12.0 Hz, 1H,1×CH₂Bn), 4.52 (m, 4H, H-1C, 3×CH₂Bn), 4.45 (m, 1H, H-6E′a), 4.39 (m,3H, 3×CH₂Bn), 4.34-4.04 (m, 15H, H-2E, H-2E′, 2×H-6E, H-6E′b, H-2A,H-2B, H-3A, H-3B, H-4A, H-4B, H-2D, H-5E′, H-6 Da, H-4D), 3.82 (m, 1H,H-5E), 3.78 (m, 1H, H-5D), 3.69 (m, 2H, H-4C, H-6Ca), 3.62-3.48 (m, 5H,H-6Aa, H-6Ba, H-6Cb, H-6Ab, H-6Db), 3.44 (dd, J=3.9, 11.3 Hz, 1H,H-6Bb), 3.31 (m, 2H, H-5B, H-3C), 3.20 (m, 1H, H-5A), 2.94 (m, 1H,H-5C), 2.19, 2.15, 2.11, 2.07, 2.04, 2.02, 1.88, 1.85 (s, 3H, CH₃ Ac).¹³C NMR (126 MHz, CDCl₃): 170.7, 170.2, 170.1, 170.0, 169.5, 167.6,138.6, 138.5, 138.3, 137.8, 137.2, 134.4, 134.3, 131.6, 131.3, 128.6,128.2, 128.1, 128.0, 127.9, 127.8, 127.5, 127.4, 127.3, 127.2, 126.8,123.7, 123.1, 98.1 (C-1D), 97.4 (C_(1E), C-1C), 97.1 (C-1A, C-1B), 95.9(C-1E′), 77.2, 76.5, 75.9, 75.3, 75.2, 74.5, 74.4, 74.3, 73.1, 72.8,71.8, 71.3, 70.7, 70.5, 70.4, 68.9, 68.8, 68.5, 68.3, 68.1, 67.1, 62.7,62.0, 61.1, 56.5, 55.7, 54.8, 54.4, 20.8, 20.7, 20.6, 20.4.

Benzyl[di-(O-3,4,6-tri-O-acetyl-2-deoxy-2-phtalimido-β-D-glucopyranosyl)-(1→2)-(1→4)-3,6-di-O-acetyl-α-D-mannopyrannosyl]-(1→3)-[O-3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl]-(1→6)-2-O-acetyl-β-D-mannopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phtalimido-β-D-glucopiranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phtalimido-β-D-glucopyranoside(20)

A suspension of acceptor 19 (75 mg, 0.03 mmol), donor 11 (41 mg, 0.05mmol, 1.5 eq, synthesized according to Unverzagt, C.; Eller, S.;Mezzato, S.; Schuberth, R. Chem. Eur. J. 2007, 14, 1304-1311) andmolecular sieves in dry DCM (9.8 mL) was stirred at room temperature for1h. The mixture was cooled to −40° C. and TfOH (1 μL, 0.01 μmol, 33%)was added. The reaction mixture was stirred at −40° C. until donor haddisappeared (1h). Then, the reaction was quenched with Et₃N, filteredover a plug of celite and concentrated. The residue was purified byflash column chromatography, obtaining the titled compound (60 mg, 62%).

[α]²⁰ _(D): +2.1 (c 0.53, CH₃C₁). ¹H NMR (500 MHz, CDCl₃): 7.88-7.61 (m,19H, H-arom), 7.30-7.23 (m, 11H, H-arom), 7.18, 7.04 (m, 1H, H-arom),7.01-6.93 (m, 7H, H-arom), 6.85, 6.75 (m, 3H, H-arom), 5.79 (dd, J=9.0,10.7 Hz, 1H, H-₃v), 5.70 (m, 2H, H-3E, H-3E″), 5.45 (d, J=8.2 Hz, 1H,H-1E′), 5.33 (d, J=8.4 Hz, 1H, H-1E), 5.24-5.09 (m, 7H, H-1E″, H-1A,H-4E′, H-4D′, H-2C, H-4E, H-4E″), 4.98. (m, 2H, H-3D, H-3D′), 4.94 (d,J=8.5 Hz, 1H, H-1B), 4.83 (d, J=12.0 Hz, 1H, 1×CH₂Bn), 4.78 (d, J=1.8Hz, 1H, H-1D), 4.71 (m, 2H, 2×CH₂Bn), 4.59 (d, J=12.0 Hz, 1H, 1×CH₂Bn),4.51-3.98 (m, 27H, H-1C, 6×CH₂Bn, 2×H-6E′, H-1D′, H-2E″, H-2E, 2×H-6E,H-2E′, H-6E″a, H-2D, H-2D′, H-2B, H-3B, H-4B, H-2A, H-3A, H-4A, H-5E′,H-6 Da, H-4D), 3.87 (m, 2H, H-6E″b, H-5E), 3.81-3.69 (m, 6H, H-5D,H-6D′a, H-4C, H-6Ca, H6Db, H5D′), 3.59-3.45 (m, 5H, 2×H-6Aa, H-6Ba,H-6D′b, H-5E″), 3.39 (dd, J=4.0, 11.5 Hz, 1H, H-6Bb), 3.33 (dd, J=5.0,10.3 Hz, 1H, H-6Cb), 3.26 (m, 2H, H-5B, H-3C), 3.17 (m, 1H, H-5A), 3.01(m, 1H H-5C), 2.15, 2.09, 2.09, 2.05, 2.04, 2.01, 2.01, 2.00, 1.95,1.89, 1.88, 1.87, 1.85, 1.85 (s, 3H, CH₃ Ac). ¹³C NMR (126 MHz, CDCl₃):170.8, 170.7, 170.6, 170.5, 170.3, 170.2, 170.1, 170.0, 169.9, 169.4,169.3, 169.2, 167.6, 167.4, 138.8, 138.6, 138.3, 138.0, 137.2, 134.3,133.4, 131.7, 131.3, 128.5, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7,127.4, 127.2, 126.8, 123.7, 123.5, 123.0, 98.6 (C-1D), 97.7 (C-1C,C-1D′), 97.3 (C-1E), 97.2 (C-1E″), 97.1 (C-1A, C-1B), 95.9 (C-1E′),77.7, 76.5, 75.8, 75.0, 74.5, 74.3, 73.0, 72.8, 72.4, 71.7, 71.2, 70.7,70.6, 70.4, 70.2, 70.0, 69.6, 69.0, 68.9, 68.8, 68.6, 68.3, 68.1, 65.5,62.7, 62.4, 61.9, 61.7, 61.5, 61.2, 56.5, 55.7, 54.9, 54.4, 20.7, 20.6,20.5, 20.4, 20.3.

[(2-acetamido-β-D-glucopyranosyl)-(1→2)-α-D-mannopyrannosyl]-(1→6)-[di-(2-acetamido-β-D-glucopyranosyl)-(1→2)-(1→4)-α-D-mannopyrannosyl]-(1→3)-β-D-mannopyranosyl-(1→4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-1,3,6-tri-O-benzyl-2-deoxy-β-D-glucopyranoside(¹³C₁₀) (22)

To a solution of compound 20 (43 mg, 14.1 μmol) in MeOH:DCM 2:1 (300:150μL), NaOMe (42 μL, 21.2 μL, 1.5 eq) was added. After stirring 1 h atroom temperature, MeOH (300 μL) and ethylenediamine (300 μL) were addedand the mixture heated for 3 cycles of 30 min at 120° C. in a microwave.The mixture was concentrated to dryness using toluene and ethanol. Thecrude residue was purified by Sephadex LH-20 column (MeOH) to givecompound 21. Compound 21 was dissolved in MeOH (200 μL) at 0° C. andacetic anhydride ¹³C_(a) was added. After 2 h, EtOH was added and themixture concentrated and purified by Sephadex LH-20 column (MeOH) togive the titled compound (11 mg, 40%, 2 steps).

¹H NMR (500 MHz, MeOD): 7.41-7.27 (m, 22H, H-arom), 7.19 (m, 3H,H-arom), 5.03 (m, 3H, H-1D, 2×CH₂Bn), 4.85 (d, J=12.0 Hz, 1H, 1×CH₂Bn),4.80 (d, J=1.9 Hz, 1H, H-1D′), 4.77 (d, J=12.0 Hz, 1H, 1×CH₂Bn),4.68-4.57 (m, 6H, H-1C, H-1B, 4×CH₂Bn), 5.50 (m, 2H, H-1E″, H-1A), 4.46(s, 2H, 2×CH₂Bn), 4.43 (d, J=8.3 Hz, 1H, H-1E), 4.32 (d, J=8.3 Hz, 1H,H-1E′), 4.13 (m, 1H, H-2D), 4.08 (m, 2H, H-3D, H-2C), 4.02 (m, 2H, H-4A,H-4B), 3.95-3.58 (m, 30H, H-2A, 2×H-6C, 2×H-6E, 2×H-6E″, 2×H-6E′,2×H-6D, 2×H-6D′, H-5D, H-2D′, H-2B, H-4C, H-3D′, 2×H-6B, 2×H-6A, H-2E,H-4D, H-3B, H-2B, H-3A, H-2E′, H-5D′, H-4D′), 3.52-3.43 (m, 5H, H-5A,H-3E, H-3E′, H-3E″, H-3C), 3.36 (m, 3H, H-5E″, H-4E′, H-4E″), 3.33 (m,2H, H-5B, H-4E), 3.26 (m, 1H, H-5E), 3.18 (m, 2H, H-5C, 2.14 (m, 4.5H,¹³CH₃ Ac), 1.97 (dd, J=6.4, 17.8 Hz, 3H, ¹³CH₃ Ac), 1.89 (m, 4.5H, ¹³CH₃Ac), 1.72 (dd, J=6.4, 17.8 Hz, 3H, ¹³CH₃ Ac). ^(‘3C) NMR (126 MHz, MeOD,HSQC): 128.2-126.6, 131.6 (C-1E″), 100.5 (C-1E, C-1E″), 100.2 (C-1A),100.0 (C-1C, C-1B), 99.6 (C-1D), 97.1 (C-1D’), 82.0, 81.0, 80.4, 78.1,77.7, 76.5, 75.6, 75.2, 74.7, 73.9, 73.1, 72.2, 70.3, 68.3, 67.6, 65.8,61.2, 55.7, 54.6, 22.3 (¹³CH₃).

Enzymatic Elongation

[β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-[β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-α,β-D-glucopyranose(15)

A solution (1 mL) of 14 (2.126 mg, 1.6 μmol), Uridine5′-diphospho-α-D-galactose disodium salt UDP-Gal (2.280 mg, 3.74 μmol,2.4 eq), bovine serum albumin BSA (1 mg), 200 mU of bovine milkβ-1,4-galactosyltransferase 2.4.4.22, 9.2 U of alkaline phosphatase3.1.3.1. and MnCl₂ (10 mM) in 770 μL Hepes buffer (50 mM, pH=7.4) wasincubated at 37° C. for 18h. The resulting mixture was heated at 95° C.for 5 min to precipitate the enzyme. After centrifugation, thesupernatant was purified through a graphite cartridge to give the titlecompound (2.09, 78%).

¹H NMR (500 MHz, D₂O) δ 5.12 (d, J=2.7 Hz, 0.6H, H-1_(α-GlcNAc)), 5.05(d, J=1.4 Hz, 1H, H-1_(α-1,3-Man)), 4.86 (d, J=1.6 Hz, 1H,H-1_(α-GlcNAc)), 4.66-4.59 (m, 0.4H, H-1_(β-GlcNAc)), 4.53 (dd, J=15.9,7.9 Hz, 3H, H-1_(β-GlcNAc)), 4.40 (dd, J=7.8, 3.1 Hz, 2H, H-1_(β-Gal)),4.18 (d, J=2.7 Hz, 1H), 4.12 (dd, J=3.3, 1.6 Hz, 1H), 4.08-4.00 (m, 1H),3.95-3.36 (m, 60H), 2.17-2.04 (m, 6H, Ac), 1.93-1.78 (m, 6H, Ac). ¹³CNMR selected peaks from HSQC experiment (126 MHz, D₂O) δ=102.9(C-1_(β-Gal)) 101.3 (H-1_(β-GlcNAc)), 100.4 (C-1_(β-Man)), 99.6(C-1_(α-1,3-Man)), 99.5 (C-1_(β-GlcNAc)), 97.1 (C-1_(α-1,6-Man)), 94.8(C-1_(β-GlcNAc)), 90.4 (C-1_(α-GlcNAc)).

[(2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-[2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-[α-L-fucopyranosyl-(1→6)]-2-acetamido-2-deoxy-α,β-D-glucopyranose(16)

A solution (1 mL) of 14 (3.030, mg, 2.28 μmol), Guanosine5′-diphospho-β-L-fucose disodium salt GDP-Fuc (1.760 mg, 2.77 μmol, 1.2eq), bovine serum albumin BSA (1 mg), a core α-1,6-fucosyltransferase(50 μL, 0.66 mg/mL) and MnCl₂ (20 mM) in 770 μL MES buffer (80 mM,pH=6.5) was incubated at room temperature for 18h. The resulting mixturewas heated at 95° C. for 5 min to precipitate the enzyme. Aftercentrifugation the supernatant was purified through a graphite cartridgeto give the title compound (2.73 mg, 82%).

¹H NMR (500 MHz, D₂O) δ 5.11 (d, J=3.2 Hz, 0.6H, H-1_(α-GlcNAc)), 5.04(d, J=1.8 Hz, 1H, H-1_(α-1,3-man)), 4.88-4.79 (m, 2H, H-1_(α-1,6-Man),H-1_(α-Fuc)), 4.64-4.55 (m, 0.4H+1H, H-1_(β-GlcNAc)), 4.48 (d, J=8.4 Hz,2H, H-1_(β-GlcNAc)), 4.18 (d, J=2.4 Hz, 1H), 4.11 (dd, J=3.3, 1.6 Hz,1H), 4.09-3.98 (m, 2H), 3.98-3.75 (m, 11H), 3.75-3.31 (m, 33H),2.25-2.04 (m, 6H, Ac), 1.95-1.78 (m, 6H, Ac), 1.14 (dd, J=6.6, 4.8 Hz,3H, CH₃ Fuc). ¹³C NMR selected from HSQC experiment (126 MHz, D₂O)δ=101.0 (C-1_(β-GlcNAc)), 100.3 (C-1_(βMan)), 99.6 (C-1_(β-GlcNAc)),99.5 (C-1_(α-1,3-Man)), 99.5 (C-1_(α-Fuc)), 97.0(C-1_(α-1,6-Man)), 94.8(C-1_(β-GlcNAc)), 90.4 (C-1_(α-GlcNAc), 15.4 (CH₃ Fuc).

[β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl)-(1→6)]-[β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl-(1→3)]-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-[α-L-fucopyranosyl-(1→6)]-2-acetamido-2-deoxy-α,β-D-glucopyranose(17)

A solution (0.5 mL) of 16 (1.836 mg, 1.6 μmol), Uridine5′-diphospho-α-D-galactose disodium salt UDP-Gal (2.210 mg, 3.62 μmol,2.9 eq), bovine serum albumin BSA (1 mg), 200 mU of bovine milkβ-1,4-galactosyltransferase 2.4.4.22, 9.2 U of alkaline phosphatase3.1.3.1. and MnCl₂ (10 mM) in 450 μL Hepes buffer (50 mM, pH=7.4) wasincubated at 37° C. for 18h. The resulting mixture was heated at 95° C.for 5 min to precipitate the enzyme. After centrifugation thesupernatant was purified through a graphite cartridge, to give the titlecompound (1.80 mg, 80%).

¹H NMR (500 MHz, D₂O) δ 5.11 (d, J=3.1 Hz, 0.6H, H-1_(α-GlcNAc)), 5.05(d, J=1.5 Hz, 1H, H-1_(α-1,3-Man)), 4.85 (s, 1H, H-1_(α-1,6-Man)), 4.82(t, J=3.7 Hz, 1H, H-1_(α-Fuc)), 4.62 (d, J=7.9 Hz, 0.4H,H-1_(β-GlcNAc)), 4.59 (d, J=7.7 Hz, 1H, H-1_(β-GlcNAc)), 4.51 (d, J=7.7Hz, 2H, H-1_(β-GlcNAc)), 4.40 (dd, J=7.8, 2.7 Hz, 2H, H-1_(β-Gal)) 4.18(s, 1H), 4.12 (d, J=3.3 Hz, 1H), 4.08-4.00 (m, 2H), 3.97-3.39 (m, 54H),2.18-2.07 (m, 6H), 1.92-1.81 (m, 6H, Ac), 1.14 (dd, J=6.6, 5.0 Hz, 3H,CH₃ Fuc).¹³C NMR peaks selected from HSQC experiment (126 MHz, D₂O)6=102.9 (H-1_(β-Gal)), 101.0 (H-1_(β-GlcNAc)), 100.4 (H-1_(β-Man)), 99.5(H-1_(β-GlcNAc)), 99.5 (H-1_(β-1,3-Man)), 99.3 (H-1_(α-Fuc)), 96.8(H-1_(α-1,6-Man)), 94.8 (H-1_(β-GlcNAc)), 90.4 (C-1_(α-GlcNAc)), 15.5(CH₃, Fuc).

Preparation of Asymmetric Mono-Galactosylated Glycan Standards

Galactosylation of ¹³C₈-G0(Bn₅)

The partially deprotected ¹³C₈-G0(Bn₅) standard 13 (1.1 mg) was treatedwith β-1,4-galactosyltransferase (200 mU) and uridine diphosphategalactose (UDP-Gal, 1.25 equivalents) in HEPES buffer 50 mM at pH 7.4,containing MnCl₂ 2 mM and BSA. After 1h of reaction at 37° C. theproteic fraction was precipitated by heating at 95° C. for 5 minutes andremoved by filtration. This solution was directly analysed by UPLC-MSshowing a 23% and 26% conversion to ¹³C₈-G1⁶(Bn₅) and ¹³C₈-G1³(Bn₅)respectively.

This reaction could be scaled up to the use of 10 mg of ¹³C₉-G0(Bn₅) asacceptor.

The reaction crude after protein precipitation from the enzymaticelongation of 10 mg of ¹³C₈-G0(Bn₅) was evaporated and then thedifferent compounds were purified by HPLC in a C₁₈ semi-preparativecolumn in reverse phase water/ACN to yield the compounds ¹³C₈-G1³(Bn₅)(2.4 mg), ¹³C₈-G1⁶(Bn₅) (2.2 mg) and ¹³C₈-G2 (Bn₅) (2.0 mg) in pure form(wherein G1³ and G1⁶ denote the respective mono-galactosylated compoundsand G2 the bis-galactosylated compound).

Both isomeric mono-galactosylated compounds were subjected tohydrogenolysis in MeOH using 1 atm of H₂ gas in an H-Cube flow reactorwith a 10% Pd/C cartridge, obtaining the ¹³C-labeled N-glycans ¹³C₈-G1³(1.2 mg) and ¹³C₈-G1⁶ (1.1 mg) in pure form.

Fucosylation of ¹³C₈-G1⁶

The compound ¹³C₈-G1⁶ (1.1 mg) was treated with a coreα-1,6-fucosyltransferase and guanosine diphosphate fucose (GDP-Fuc, 1.10equivalents) in MES buffer 50 mM at pH 6.5, containing MgCl₂ 2 mM. After18h of reaction at 30° C. the proteic fraction was precipitated byheating at 95° C. for 5 minutes and filtered off. The glycan ¹³C₈-G1⁶Fwas purified with a graphitized carbon cartridge.

Galactosylation of ¹³C₈-G0(Bn₅)

The partially deprotected ¹³C₈-G0(Bn₅) standard (20 μg) was treated withβ-1,4-galactosyltransferase (200 mU) and uridine diphosphate galactose(UDP-Gal, 6.0 equivalents) in HEPES buffer 50 mM at pH 7.4, containingMnCl₂ 2 mM and BSA. After 1h of reaction at 37° C. the proteic fractionwas precipitated by heating at 95° C. for 5 minutes and filtered off.

This solution was directly analysed by UPLC-MS' showing completeconversion to ¹³C₈-G2(Bn₅).

The bis-galactosylated compound ¹³C₈-G2(Bn₅) (20 ug) was treated withβ-1,4-galactosidase from A. oryzae (15 mU) in phosphate/citrate buffer50 mM at pH 4.5 for 18h at 30° C. and the reaction was quenched by theaddition of MeOH (20 μL). This solution was directly analysed by UPLC-MSshowing conversions of 10% and 46% to the mono-galactosylated compounds¹³C₈-G1⁶(Bn₅) and ¹³C₈-G1³(Bn₅) respectively.

Preparation of Asymmetric α-2,3-Silylated Glycan Standards

Silylation of ¹³C₈-G2(Bn₅)

The reaction was performed at analytical scale from the previouslyprepared bis-galactosylated biantennary ¹³C₈-G2(Bn₅) (10 nmol). Thiscompound was treated with 10 mU of α-2,3-sialyltransferase from Pmultocida and cytidine monophosphate N-acetylneuraminic acid(CMP-NeuNAc, 4 equivalents) in Tris.HCl buffer 100 mM at pH 8.0,containing 20 mM MgCl₂ at 37° C. for 30 minutes. The reaction wasquenched by the addition of MeOH (20 μL). This solution was directlyanalysed by UPLC-MS showing a 46% conversion to the mono-sialylatedcompound ¹³C₈-G2A1(Bn₅), separated into two isomeric peaks (these beingthe corresponding 3- and 6-mono-silylated products), and a 32%conversion to the bis-sialylated compound ¹³C₈-G2A2(Bn₅).

The reaction was performed at preparative scale from the previouslyprepared bis-galactosylated biantennary ¹³C₈-G2(Bn₅). A solution (100μL) of ¹³C₈-G2Bn₅ (1.0 mg, 0.48 μmol),Cytidine-5′-monophospho-N-acetylneuraminic acid sodium salt CMP-NeuAc(0.72 mg, 0.96 μmol, 2 eq), 100mU of α-2,3-Sialyltransferase fromPasteurella multocida 2.4.99.4 and MgCl₂ (100 mM) in 500 μL Tris-HClbuffer (1M, pH=8) was incubated at 37° C. for 30 min. MeOH (500 μL) wasadded to the resulting mixture to precipitate the enzyme. Aftercentrifugation, the supernatant was purified by HPLC in a C18semipreparative column in reverse phase water/ACN to give three newisotopically-labelled glycan standards, the two ¹³C₈-G2A1(Bn₅) compoundsand ¹³C₈-G2A2(Bn₅).

Synthesis of ¹³C₈-G1A1³Bn₅

A solution (100 μL) of ¹³C₈-G1³(Bn₅) (1.0 mg, 0.52 μmol),Cytidine-5′-monophospho-N-acetylneuraminic acid sodium salt CMP-NeuAc(0.78 mg, 1.04 μmol, 2 eq), 100mU of α-2,3-Sialyltransferase fromPasteurella multocida 2.4.99.4 and MgCl₂ (100 mM) in 500 μL Tris-HClbuffer (1M, pH=8) was incubated at 37° C. for 30 min. MeOH (500 μL) wasadded to the resulting mixture to precipitate the enzyme. Aftercentrifugation, the supernatant was purified by HPLC in a C₁₈semipreparative column in reverse phase water/ACN obtaining compound¹³C₈-G1A1³(Bn₅) (0.68 mg, 59% Yield).

Preparation of α-2,6-Silylated Glycan Standards

The reaction was performed at analytical scale from the previouslyprepared bis-galactosylated biantennary ¹³C₈-G2(Bn₅) (5 nmol). Thiscompound was treated with 0.25mU of human α-2,6-sialyltransferase andcytidine monophosphate N-acetylneuraminic acid (CMP-NeuNAc, 1-4equivalents) in cacodylate buffer 50 mM at pH 6.1, containing 2 mM MnCl₂at 37° C. for 2-4 hours. The reaction was quenched by the addition ofMeOH (20 μL). This solution was directly analysed by UPLC-MS and theresults are presented in Table 3.

Preparation of Truncated N-Glycan Standards

¹³C₈-G0(Bn₅) (3 mg) was treated with 100 mU of N-acetyl glucosaminidasefrom Conavalia ensiformis in ammonium acetate buffer 50 mM at pH 4.5 atr.t. for 6h. The reaction was quenched by the addition of MeOH (20 μL).This solution was directly analysed by UPLC-MS obtaining conversions of20%, 26% and 25% of ¹³C₆-MGn³(Bn₅), ¹³C₆-MGn⁶(Bn₅) and ¹³C₄-Man3(Bn₅)respectively. After semipreparative HPLC purification the pure compounds¹³C₆-MGn³(Bn₅) (2.2 mg), ¹³C₆-MGn⁶(Bn₅) (2.0 mg) and ¹³C₄-Man3(Bn₅) (1.7mg) were obtained.

The three isolated compounds were subjected to hydrogenolysis in MeOHusing 1 atm of H2 gas in an H-Cube flow reactor with a 10% Pd/Ccartridge, obtaining the ¹³C-labeled N-glycans ¹³C₆-MGn³ (1.2 mg) and¹³C₆-MGn⁶ (1.1 mg) and ¹³C₄-Man3 (0.7 mg) in pure form.

Fucosylation of ¹³C₆-MGn³

The compound ¹³C₆-MGn³ (1.2 mg) was treated with a coreα-1,6-fucosyltransferase and guanosine diphosphate fucose (GDP-Fuc, 1.10equivalents) in MES buffer 50 mM at pH 6.5, containing MgCl₂ 2 mM. After18h of reaction at 30° C. the proteic fraction was precipitated byheating at 95° C. for 5 minutes and filtered off. The glycan ¹³C₆-MGn³Fwas purified with a graphitized carbon cartridge.

Galactosylation of Partially-Protected Triantennary Core 22

A solution (24 μL) of triantennary 22 (120 μg, 0.06 μmol), Uridine5′-diphospho-α-D-galactose disodium salt UDP-Gal (55 μg, 0.09 μmol, 1.5eq), 24mU of bovine milk β-1,4-galactosyltransferase 2.4.4.22 and MnCl₂(10 mM) in 300 μL HEPES buffer (50 mM, pH=7.4) was incubated at 37° C.for 1 h. The resulting mixture was heated at 95° C. for 5 min toprecipitate the enzyme. After centrifugation, the supernatant wasdirectly analysed by UPLC-MS. All seven possible galactosylated productswere detected (tris, all three bis and all three mono), in addition tothe non-galactosylated starting material.

REFERENCES

All publications, patents and patent applications cited herein or filedwith this application, including references filed as part of anInformation Disclosure Statement, are incorporated by reference in theirentirety.

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The invention claimed is:
 1. A glycan comprising the motif:

wherein each Ac* is isotopically-labelled.
 2. The glycan of claim 1,wherein each Ac* is selected from —(¹³C═O)¹³CH₃, —(C═O)¹³CH₃,—(¹³C═O)CH₃, —(C═O)CD₃, —(¹³C═O)¹³CD₃, —(C═O)¹³CD₃, —(¹³C═O)CD₃,—(¹⁴C═O)¹⁴CH₃, —(C═O)¹⁴CH₃, —(¹⁴C═O)CH₃, —(C═¹⁷O) CH₃, —(¹³C═¹⁷O) CH₃,—(C═¹⁷O)¹³CH₃, —(¹³C═¹⁷O)¹³CH₃, —(C═¹⁸O)CH₃, —(¹³C═¹⁸O) CH₃,—(C═¹⁸O)¹³CH₃, —(¹³C═¹⁸O)¹³CH₃.
 3. The glycan according to claim 2,wherein the glycan comprises one or more further monosaccharide units.4. A kit for identifying a glycan in a sample, the kit comprising: (a) atagged standard, the tagged standard comprising an isotopically-labelledglycans according to claim 1

(b) instructions for doping a sample suspected of containing a glycanwith the tagged standard to obtain a doped sample and analysing thedoped sample using mass spectrometry.
 5. The kit of claim 4, wherein theinstructions include mass spectrometry data for the tagged standard. 6.The kit of claim 4, wherein the instructions include the step ofcomparing the ion peaks associated with the tagged standard with theadditional ion peaks in the mass spectrum.
 7. A method for the synthesisof an isotopically-labelled glycan as claimed in claim 1 for use as amass spectrometry internal standard, the method comprising: acylating anoligosaccharide core structure with an isotopically-labelled acylatingagent, wherein the oligosaccharide core structure is optionallyprotected with one or more protecting groups, to obtain anisotopically-labelled oligosaccharide core structure; and enzymaticallyderivatising the resultant isotopically-labelled oligosaccharide toobtain the isotopically-labelled glycan.
 8. The method of claim 7,wherein the enzymatic derivatisation comprises an enzymatic hydrolysisstep to remove a terminal sugar unit.
 9. The method of claim 7, whereinthe enzymatic derivatisation comprises an enzymatic elongation step witha glycosyltransferase and a suitable sugar donor, optionally wherein theenzymatic elongation step incorporates a sugar unit that is itselfisotopically-labelled.
 10. The method of claim 7, wherein theisotopically-labelled oligosaccharide core structure is protected withone or more protecting groups during enzymatic derivatisation.
 11. Themethod of claim 10, wherein the isotopically-labelled oligosaccharidecore structure is protected with one or more optionally substitutedbenzyl groups.
 12. The method of claim 7, wherein the oligosaccharidecore structure comprises a disaccharide motif, the disaccharide motifcomprising a first monosaccharide unit and a second monosaccharide unit,wherein at least one of the first monosaccharide unit and/or secondmonosaccharide unit comprises an amino group and acylation occurs at theamino group(s).
 13. The method according to claim 7, wherein theisotopically-labelled acylating agent is isotopically-labelled aceticanhydride.
 14. The method according claim 13, wherein theisotopically-labelled acetylating agent is selected from: (¹³CH₃¹³C═O)₂, (¹³CH₃C═O)₂, (CH₃ ¹³C═O)₂, (CD₃C═O)₂, (¹³CD₃ ¹³C═O)₂,(¹³CD₃C═O)₂ or (CD₃ ¹³C═O)₂.
 15. The method of claim 7, wherein themethod further comprises forming an oxazoline at a free anomericposition of an acetyl-hexosamine unit in the isotopically-labelledoligosaccharide.
 16. The method of claim 7, wherein the method furthercomprises glycosylating a peptide, lipid or protein to obtain anisotopically-labelled glycopeptide, peptidoglycan, glycolipid,glycoprotein comprising the isotopically-labelled oligosaccharide.
 17. Amethod comprising: (i) obtaining a sample suspected of containing aglycan associated with a disease or disorder from a patient; (ii)selecting a tagged standard that comprises an isotopically-labelledglycan as claimed in claim 1

and corresponds to a glycan associated with the disease or disorder;(iii) adding the tagged standard to the sample to obtain a doped sample;(iv) analyzing the doped sample using mass spectrometry to obtain ionpeaks; (v) comparing the identity and intensity of the ion peaksassociated with the tagged standard with the additional ion peaks in thespectrum of the doped sample to identify, and optionally to quantify,the presence of one or more glycans in the sample; (vi) using thepresence of said one or more glycans to diagnose the disease ordisorder.
 18. The method of claim 17, wherein the medical disease ordisorder is selected from cancer, a cardiovascular disorder, aninflammatory skin disease, diabetes mellitus, a gastrointestinaldisorder, a liver disorder, anaemia, an immunological disease ordisorder, autoimmune disease, arthritis, including rheumatoid arthritis,an infectious disease, nephropathy, a neurological disorder, a pulmonarydisorder or a congenital disorder of glycosylation.